Validation of short culture method for rapid bacterial identification of blood cultures via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

BACKGROUND: Development of rapid bacterial identification from blood cultures has been an area of intense study in diagnostic microbiology. Shortened turnaround time coupled with antimicrobial stewardship interventions have been shown to improve patient outcomes and decrease healthcare-associated costs. OBJECTIVES: We report the validation of a short incubation method for Gram-positive and Gram-negative bacterial identification utilizing MALDI-TOF MS without additional instrumentation, processing or cost compared with current practice. METHODS: Prospective, observational, single-centre study in a quaternary care academic hospital encompassing 376 blood cultures subjected to bacterial identification after short incubation periods of 3-4 and 6-8 h. RESULTS: There was 97.5% species-level identification agreement with tests undertaken after 3-4 h incubation with 83.6% isolates identified, and 99.7% species-level identification agreement after 6-8 h incubation with 96.7% isolates identified. CONCLUSIONS: The short incubation method provides a rapid MALDI-TOF MS bacterial identification method, reducing turnaround time by 10-18 h compared with standard practice without additional cost, processing or instrumentation.

Prevalence and associations of frailty in residents of Australian aged care facilities: findings from a retrospective cohort study.

OBJECTIVES: Studies conducted among older people have shown that frailty is a common condition associated with an array of adverse outcomes. The aims of this study were to identify the prevalence and associations of frailty in older people residing in several aged care facilities located in Queensland, Australia. METHODS: The database used for this study was drawn from the Aged Care Funding Instrument (ACFI) database of an Australian aged care provider, and contained data from ten aged care facilities in Queensland, Australia. A modification of an eFI originally developed by Clegg and colleagues and based on Rockwood's Frailty Index (FI) of cumulative deficits was used to identify frailty. RESULTS: In total, 592 participants aged 75 years and over were included in the study (66.6% female). Median (IQR) age was 88.0 (9.0) years. Frailty prevalence among the sample was 43.6%, with 46.3% pre-frail and 10.1% not frail. In a multivariate logistic regression analysis incorporating three different models, frailty was significantly associated with three ACFI domains (Nutrition, Depression and Complex Health Care), along with facility size, consistently across two models. In the third model, frailty was also significantly associated with arthritis, diabetes, hypertension, osteoporosis and vision problems, along with male gender. CONCLUSION: There is a need to develop frailty identification and management programs as part of standard care pathways for older adults residing in aged care facilities. Aged care facilities should consider regular frailty screening in residential aged care residents, along with interventions addressing specific issues such as dysphagia and depression.

Epidemiology of viral respiratory infections in Australian working-age adults (20-64 years): 2010-2013.

Acute respiratory infections cause significant morbidity and mortality accounting for 5.8 million deaths worldwide. In Australia, influenza-like illness (ILI), defined as cough, fever and fatigue is a common presentation in general practice and results in reduced productivity and lost working days. Little is known about the epidemiology of ILI in working-age adults. Using data from the ASPREN influenza surveillance network in Australia (2010-2013) we found that working-age adults made up 45.2% of all ILI notifications with 55% of samples positive for at least one respiratory virus. Viruses most commonly detected in our study included influenza A (20.6%), rhinovirus (18.6%), influenza B (6.2%), human meta-pneumovirus (3.4%), respiratory syncytial virus (3.1%), para-influenza virus (2.6%) and adenovirus (1.3%). We also demonstrated that influenza A is the predominant virus that increases ILI (by 1.2% per month for every positive influenza A case) in working-age adults during autumn-winter months while other viruses are active throughout the year. Understanding the epidemiology of viral respiratory infections through a year will help clinicians make informed decisions about testing, antibiotic and antiviral prescribing and when the beginning of the 'flu season' can be more confidently predicted.

Nutritional intervention as part of functional rehabilitation in older people with reduced functional ability: a systematic review and meta-analysis of randomised controlled studies.

BACKGROUND: Nutritional intervention is increasingly recognised as having an important role in functional rehabilitation for older people. Nonetheless, a greater understanding of the functional benefit of nutritional interventions is needed. METHODS: A systematic review and meta-analysis examined randomised controlled trials (RCTs) published between 2007 and 2014 with the aim of determining whether nutritional intervention combined with rehabilitation benefited older people with reduced functional ability. Six electronic databases were searched. RCTs including people aged 65 years and older with reduced physical, social and/or cognitive function were included. PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines were followed, and gradepro computer software (http://gradepro.org) was used for the quality assessment of critical and important outcomes. Included studies considered to be clinical homogenous were combined in a meta-analysis. RESULTS: Of the 788 studies screened, five were identified for inclusion. Nutritional intervention given with functional rehabilitation improved energy and protein intake, although it failed to provide any improvement in final body weight, hand-grip strength or muscle strength. There was no difference between groups in the critical outcomes; balance, cognition, activities of daily living and mortality at long-term follow-up. Nutritional intervention given with functional rehabilitation was associated with an increased likelihood of both mortality (odds ratio = 1.77; 95% confidence interval = 1.13-2.76) and hospitalisation (odds ratio = 2.29; 95% confidence interval = 1.10-4.79) during the intervention. Meta-analysis of the baseline data showed that, overall, the intervention cohort had a lower body weight and cognition. CONCLUSIONS: This meta-analysis highlights concerns regarding the quality of the randomisation of participants at baseline. Future high-quality research is essential to establish whether older people with loss of functional abilities can benefit from nutritional intervention.

Thrombin generation and coagulation factor content of thawed plasma and platelet concentrates.

BACKGROUND: We assessed the haemostatic capacity of thawed plasma produced after ambient storage of whole blood for 24 h (RTFP24), and the supernatant of buffy-coat derived platelet concentrates (PC). METHODS: Platelet concentrates (n = 20) were tested on days 1, 5 and 7 of storage at 22°C and RTFP24 (n = 10) immediately following thawing and after 4 and 6 days storage at 4°C. Coagulation factor activity, thrombin generation ± an activator of protein C (PROTAC) and rotational thromboelastometry (ROTEM) were assessed. RESULTS: In plasma and buffy-coat derived PC, there was a < 10% loss of factors II, IX and FX, but much higher loss of factors FV, FVII and FVIII. In plasma, the total or peak amount of thrombin generated was unaffected by storage for 6 days, with or without Protac, but there was an increase in lag time and decreased rate of clot formation by ROTEM. In PC, but not plasma, there was a 16% increase in FXII activity and increase in resistance to activated protein C, co-incidental to 30% loss of free protein S. CONCLUSIONS: These data suggest thrombin generation is relatively unaltered when RTFP24 is thawed and stored for 6 days, and that the supernatant of PC has significant haemostatic capacity.

Prognostic accuracy of eight frailty instruments for all-cause mortality in Australian primary care.

AIM: To investigate the ability of eight frailty instruments to accurately predict all-cause mortality and other adverse outcomes in Australian primary care patients. METHODS: Study participants included adults aged ≥75 years attending one of three primary care clinics in South Australia. Frailty instruments studied were Fried's frailty phenotype (FFP), the Frailty Index (FI) of cumulative deficits, Kihon Checklist (KCL), the Fatigue Resistance Ambulation Illness and Loss of weight (FRAIL) scale, Groningen Frailty Indicator (GFI), PRISMA-7, Reported Edmonton Frail Scale (REFS), and gait speed. Primary outcomes were all-cause mortality at 12- and 24-months. Secondary outcomes included falls, general practice attendance, hospital admission and emergency department (ED) presentation at 12-months. RESULTS: 243 participants (55.6 % female) with a mean (SD) age of 80.2 (4.6) years were included. 29 participants (16.6 %) were classified as frail at baseline by FFP. All frailty instruments demonstrated a significant ability to predict 12- and 24-month mortality. The REFS showed the highest auROC for both 12- and 24-month mortality. The REFS, Frailty Index, Kihon Checklist, FRAIL scale, and gait speed showed excellent discriminative ability for 12-month mortality (auROC ≥ 0.8 - >0.9), while the remainder showed acceptable discrimination. All frailty instruments, with the exception of the GFI, showed an excellent discriminative ability for 24-month mortality (auROC 0.8-<0.9). CONCLUSIONS: All frailty instruments possessed adequate discriminative ability for all-cause mortality predicting in older primary care patients. Frailty measurement is thus a valuable strategy to identify older patients at risk of mortality and can guide clinical decision-making in primary care settings.

Screening, Diagnosis and Management of Sarcopenia and Frailty in Hospitalized Older Adults: Recommendations from the Australian and New Zealand Society for Sarcopenia and Frailty Research (ANZSSFR) Expert Working Group.

Sarcopenia and frailty are highly prevalent conditions in older hospitalized patients, which are associated with a myriad of adverse clinical outcomes. This paper, prepared by a multidisciplinary expert working group from the Australian and New Zealand Society for Sarcopenia and Frailty Research (ANZSSFR), provides an up-to-date overview of current evidence and recommendations based on a narrative review of the literature for the screening, diagnosis, and management of sarcopenia and frailty in older patients within the hospital setting. It also includes suggestions on potential pathways to implement change to encourage widespread adoption of these evidence-informed recommendations within hospital settings. The expert working group concluded there was insufficient evidence to support any specific screening tool for sarcopenia and recommends an assessment of probable sarcopenia/sarcopenia using established criteria for all older (≥65 years) hospitalized patients or in younger patients with conditions (e.g., comorbidities) that may increase their risk of sarcopenia. Diagnosis of probable sarcopenia should be based on an assessment of low muscle strength (grip strength or five times sit-to-stand) with sarcopenia diagnosis including low muscle mass quantified from dual energy X-ray absorptiometry, bioelectrical impedance analysis or in the absence of diagnostic devices, calf circumference as a proxy measure. Severe sarcopenia is represented by the addition of impaired physical performance (slow gait speed). All patients with probable sarcopenia or sarcopenia should be investigated for causes (e.g., chronic/acute disease or malnutrition), and treated accordingly. For frailty, it is recommended that all hospitalized patients aged 70 years and older be screened using a validated tool [Clinical Frailty Scale (CFS), Hospital Frailty Risk Score, the FRAIL scale or the Frailty Index]. Patients screened as positive for frailty should undergo further clinical assessment using the Frailty Phenotype, Frailty Index or information collected from a Comprehensive Geriatric Assessment (CGA). All patients identified as frail should receive follow up by a health practitioner(s) for an individualized care plan. To treat older hospitalized patients with probable sarcopenia, sarcopenia, or frailty, it is recommended that a structured and supervised multi-component exercise program incorporating elements of resistance (muscle strengthening), challenging balance, and functional mobility training be prescribed as early as possible combined with nutritional support to optimize energy and protein intake and correct any deficiencies. There is insufficient evidence to recommend pharmacological agents for the treatment of sarcopenia or frailty. Finally, to facilitate integration of these recommendations into hospital settings organization-wide approaches are needed, with the Spread and Sustain framework recommended to facilitate organizational culture change, with the help of 'champions' to drive these changes. A multidisciplinary team approach incorporating awareness and education initiatives for healthcare professionals is recommended to ensure that screening, diagnosis and management approaches for sarcopenia and frailty are embedded and sustained within hospital settings. Finally, patients and caregivers' education should be integrated into the care pathway to facilitate adherence to prescribed management approaches for sarcopenia and frailty.

Repeatability of subjective evaluation of lameness in horses.

REASONS FOR PERFORMING STUDY: Previous studies have suggested that agreement between equine veterinarians subjectively evaluating lameness in horses is low. These studies were limited to small numbers of horses, evaluating movement on the treadmill or to evaluating previously-recorded videotape. OBJECTIVES: To estimate agreement between equine practitioners performing lameness evaluations in horses in the live, over ground setting. METHODS: 131 mature horses were evaluated for lameness by 2-5 clinicians (mean 3.2) with a weighted-average of 18.7 years of experience. Clinicians graded each limb using the AAEP lameness scale by first watching the horse trot in a straight line only and then after full lameness evaluation. Agreement was estimated by calculation of Fleiss' (kappa). Evaluators agreed if they picked the same limb as lame or not lame regardless of the severity of perceived lameness. RESULTS: After only evaluating the horse trot in a straight line clinicians agreed whether a limb was lame or not 76.6% of the time (kappa= 0.44). After full lameness evaluation clinicians agreed whether a limb was lame or not 72.9% of the time (kappa= 0.45). Agreement on forelimb lameness was slightly higher than on hindlimb lameness. When the mean AAEP lameness score was >1.5 clinicians agreed whether or not a limb was lame 93.1% of the time (kappa= 0.86), but when the mean score was < or = 1.5 they agreed 61.9% (kappa= 0.23) of the time. When given the task of picking whether or not the horse was lame and picking the worst limb after full lameness evaluation, clinicians agreed 51.6% (kappa= 0.37) of the time. CONCLUSIONS: For horses with mild lameness subjective evaluation of lameness is not very reliable. POTENTIAL RELEVANCE: A search for and the development of more objective and reliable methods of lameness evaluation is justified and should be encouraged and supported.

The application of artificial intelligence (AI) techniques to identify frailty within a residential aged care administrative data set.

INTRODUCTION: Research has shown that frailty, a geriatric syndrome associated with an increased risk of negative outcomes for older people, is highly prevalent among residents of residential aged care facilities (also called long term care facilities or nursing homes). However, progress on effective identification of frailty within residential care remains at an early stage, necessitating the development of new methods for accurate and efficient screening. OBJECTIVES: We aimed to determine the effectiveness of artificial intelligence (AI) algorithms in accurately identifying frailty among residents aged 75 years and over in comparison with a calculated electronic Frailty Index (eFI) based on a routinely-collected residential aged care administrative data set drawn from 10 residential care facilities located in Queensland, Australia. A secondary objective included the identification of best-performing candidate algorithms. METHODS: We designed a frailty prediction system based on the eFI identification of frailty, allocating 84.5 % and 15.5 % of the data to training and test data sets respectively. We compared the performance of 18 specific scenarios to predict frailty against eFI based on unique combinations of three ML algorithms (support vector machines [SVM], decision trees [DT] and K-nearest neighbours [KNN]) and six cases (6, 10, 11, 14, 39 and 70 input variables). We calculated accuracy, percentage positive and negative agreement, sensitivity, specificity, Cohen's kappa and Prevalence- and Bias- Adjusted Kappa (PABAK), table frequencies and positive and negative predictive values. RESULTS: Of 592 eligible resident records, 500 were allocated to the training set and 92 to the test set. Three scenarios (10, 11 and 70 input variables), all based on SVM algorithm, returned overall accuracy above 75 %. CONCLUSIONS: There is some potential for AI techniques to contribute towards better frailty identification within residential care. However, potential benefits will need to be weighed against administrative burden, data quality concerns and presence of potential bias.

Malnutrition Screening and Assessment in Hospitalised Older People: a Review.

Malnutrition (undernutrition) remains one of the most serious health problems for older people worldwide. Many factors contribute to malnutrition in older people, including: loss of appetite, polypharmacy, dementia, frailty, poor dentition, swallowing difficulties, social isolation, and poverty. Malnutrition is common in the hospital setting, yet often remains undetected by medical staff. The objective of this review is to compare the validity and reliability of Nutritional Screening Tools (NSTs) for older adults in the hospital setting. We also provide an overview of the various nutritional screening and assessment tools used to identify malnutrition in hospitalised older adults. These include: Subjective Global Assessment (SGA), the Mini Nutritional Assessment (MNA), MNA-short form (MNA-SF), Malnutrition Universal Screening Tool (MUST), Simplified Nutritional Appetite Questionnaire (SNAQ), Geriatric Nutrition Risk Index (GNRI) and anthropometric measurements. The prevalence and outcomes of malnutrition in hospitalised older adults are also addressed.

Physical Frailty: ICFSR International Clinical Practice Guidelines for Identification and Management.

OBJECTIVE: The task force of the International Conference of Frailty and Sarcopenia Research (ICFSR) developed these clinical practice guidelines to overview the current evidence-base and to provide recommendations for the identification and management of frailty in older adults. METHODS: These recommendations were formed using the GRADE approach, which ranked the strength and certainty (quality) of the supporting evidence behind each recommendation. Where the evidence-base was limited or of low quality, Consensus Based Recommendations (CBRs) were formulated. The recommendations focus on the clinical and practical aspects of care for older people with frailty, and promote person-centred care. Recommendations for Screening and Assessment: The task force recommends that health practitioners case identify/screen all older adults for frailty using a validated instrument suitable for the specific setting or context (strong recommendation). Ideally, the screening instrument should exclude disability as part of the screening process. For individuals screened as positive for frailty, a more comprehensive clinical assessment should be performed to identify signs and underlying mechanisms of frailty (strong recommendation). Recommendations for Management: A comprehensive care plan for frailty should address polypharmacy (whether rational or nonrational), the management of sarcopenia, the treatable causes of weight loss, and the causes of exhaustion (depression, anaemia, hypotension, hypothyroidism, and B12 deficiency) (strong recommendation). All persons with frailty should receive social support as needed to address unmet needs and encourage adherence to a comprehensive care plan (strong recommendation). First-line therapy for the management of frailty should include a multi-component physical activity programme with a resistance-based training component (strong recommendation). Protein/caloric supplementation is recommended when weight loss or undernutrition are present (conditional recommendation). No recommendation was given for systematic additional therapies such as cognitive therapy, problem-solving therapy, vitamin D supplementation, and hormone-based treatment. Pharmacological treatment as presently available is not recommended therapy for the treatment of frailty.

An examination of the initial cancer consultation of medical and radiation oncologists using the Cancode interaction analysis system.

This study provides an analysis of the structure of the initial cancer consultation, the consultation styles of medical and radiation oncologists, and their effect on patient outcomes. One hundred and fifty-five cancer patients attending their first consultation with either a medical or radiation oncologist were audiotaped and the transcripts were analysed using the Cancode computer interaction analysis system. Findings revealed that medical oncologists allowed patients and their families more input into the consultation and were rated as warmer and more patient-centred compared with radiation oncologists. However, radiation oncologists spent a longer period discussing, and were more likely to bring up, social support issues with patients. Both medical and radiation oncologists varied their consultation style according to the patient's gender, age, anxiety levels, prognosis, and education. Patients seeing an oncologist who was rated as warmer and discussed a greater number of psychosocial issues had better psychological adjustment and reduced anxiety after consultation. These findings provide current evidence that may be used to inform improvements of communication skills training for oncologists and highlight the need for future communication research to separately consider oncologists from different disciplines.

Socioeconomic Position and Malnutrition among Older Adults: Results from the FRADEA Study.

OBJECTIVES: Low socioeconomic position (SEP) is related to many health-related conditions in older adults. However, there is a lack of knowledge on the association between SEP and malnutrition, a condition with serious consequences for older people in terms of quality of life and adverse health events. In the current study, we investigated socioeconomic inequalities in malnutrition and sub-domains of malnutrition in a sample of Spanish older adults. DESIGN: Cross-sectional population-based study. SETTING: Urban area of Albacete, Spain. PARTICIPANTS: 836 participants over age 70 from the first measurement wave (2007-2009) of the Frailty and Dependence in Albacete (FRADEA) study, a population-based cohort study. MEASUREMENTS: Educational level and occupational level were the indicators of SEP. Nutritional risk was measured with the Mini Nutrition Assessment® Short Form (MNA®-SF). Logistic regression analyses were performed. RESULTS: For both socioeconomic indicators there was a statistically significant association with nutritional risk (OR low education=1.99, 95% CI=1.18-3.35; OR low occupational level=1.71, 95% CI=1.08-2.72). However, these associations disappeared after adjusting for age and sex (OR low education=1.51, 95% CI=0.88-2.60 ; OR low occupational level=1.32, 95% CI=0.80-2.17). In adjusted models, statistically significant associations between SEP and sub-domains of the MNA®-SF were observed, but these associations were not consistent across socioeconomic indicators. CONCLUSIONS: This study found that malnutrition is a condition that can appear in any older adult, regardless of their socioeconomic group. These findings suggest that interventions to prevent malnutrition in older adults can be targeted at a general older population, and do not have to be SEP specific.

Older People Who Are Frequent Users of Acute Care: A Symptom of Fragmented Care? A Case Series Report on Patients' Pathways of Care.

Older frequent users of acute care can experience fragmented care. There is a need to understand the issues in a local context before attempting to address fragmented care. 0.5% (n=61) of the population in a defined local government area were identified as having ≥4 unplanned emergency department (ED) presentations/ admissions to an acute-care hospital over 13 months. A retrospective case-series study was conducted to examine detailed pathways of care for 17 patients within the identified population. The two dominant presentation reasons were clinical symptoms associated with a declining/significant loss of capacity in fundamental self-care activities and chronic cardiac/respiratory conditions. Of patients discharged home, 21% of discharge letters were delayed >7 days and only 19% received a written discharge plan. Half of community dwelling patients received home nursing and/or assistance. Frequent users of acute care can experience untimely hospital communication and may require more coordinated care provided in the community to assist self-care and manage chronic conditions.

International Clinical Practice Guidelines for Sarcopenia (ICFSR): Screening, Diagnosis and Management.

OBJECTIVES: Sarcopenia, defined as an age-associated loss of skeletal muscle function and muscle mass, occurs in approximately 6 - 22 % of older adults. This paper presents evidence-based clinical practice guidelines for screening, diagnosis and management of sarcopenia from the task force of the International Conference on Sarcopenia and Frailty Research (ICSFR). METHODS: To develop the guidelines, we drew upon the best available evidence from two systematic reviews paired with consensus statements by international working groups on sarcopenia. Eight topics were selected for the recommendations: (i) defining sarcopenia; (ii) screening and diagnosis; (iii) physical activity prescription; (iv) protein supplementation; (v) vitamin D supplementation; (vi) anabolic hormone prescription; (vii) medications under development; and (viii) research. The ICSFR task force evaluated the evidence behind each topic including the quality of evidence, the benefit-harm balance of treatment, patient preferences/values, and cost-effectiveness. Recommendations were graded as either strong or conditional (weak) as per the GRADE (Grading of Recommendations Assessment, Development and Evaluation) approach. Consensus was achieved via one face-to-face workshop and a modified Delphi process. RECOMMENDATIONS: We make a conditional recommendation for the use of an internationally accepted measurement tool for the diagnosis of sarcopenia including the EWGSOP and FNIH definitions, and advocate for rapid screening using gait speed or the SARC-F. To treat sarcopenia, we strongly recommend the prescription of resistance-based physical activity, and conditionally recommend protein supplementation/a protein-rich diet. No recommendation is given for Vitamin D supplementation or for anabolic hormone prescription. There is a lack of robust evidence to assess the strength of other treatment options.

Management of Musculoskeletal Conditions in Rural South Australia: A Randomised Controlled Trial.

With age, the prevalence of musculoskeletal conditions increases markedly. This rural-based study determined the benefits of two approaches for managing musculoskeletal conditions: a multiple-component 'Self-management Plus' intervention, and usual care. The intervention combined self-management education with physical activity and health professional support. 6-month outcomes included: Clinical Global Impression-Improvement Scale (CGI-IS) and Quality of Life (QoL). A total of 145 people were recruited; mean (SD) age was 66.1 (11.1) and 63.3 (10.9) years for intervention and control groups respectively. The intervention resulted in greater improvements in global functioning (CGI-IS mean (SD) = 3.2 (1.3)) than usual care (CGI-IS mean (SD) = 4.2 (1.5)). There was no difference in QoL improvement between study groups. A multiple-component 'Self-management Plus' intervention had a positive effect on physical functioning for older adults with musculoskeletal conditions. However, recruitment and retention of participants was problematic, which raises questions about the intervention's feasibility in its current form.

Axon branching requires interactions between dynamic microtubules and actin filaments.

Cortical neurons innervate many of their targets by collateral axon branching, which requires local reorganization of the cytoskeleton. We coinjected cortical neurons with fluorescently labeled tubulin and phalloidin and used fluorescence time-lapse imaging to analyze interactions between microtubules and actin filaments (F-actin) in cortical growth cones and axons undergoing branching. In growth cones and at axon branch points, splaying of looped or bundled microtubules is accompanied by focal accumulation of F-actin. Dynamic microtubules colocalize with F-actin in transition regions of growth cones and at axon branch points. In contrast, F-actin is excluded from the central region of the growth cone and the axon shaft, which contains stable microtubules. Interactions between dynamic microtubules and dynamic actin filaments involve their coordinated polymerization and depolymerization. Application of drugs that attenuate either microtubule or F-actin dynamics also inhibits polymerization of the other cytoskeletal element. Importantly, inhibition of microtubule or F-actin dynamics prevents axon branching but not axon elongation. However, these treatments do cause undirected axon outgrowth. These results suggest that interactions between dynamic microtubules and actin filaments are required for axon branching and directed axon outgrowth.

Fibroblast growth factor-2 promotes axon branching of cortical neurons by influencing morphology and behavior of the primary growth cone.

Interstitial branching is an important mechanism for target innervation in the developing CNS. A previous study of cortical neurons in vitro showed that the terminal growth cone pauses and enlarges in regions from which interstitial axon branches later develop (Szebenyi et al., 1998). In the present study, we investigated how target-derived signals affect the morphology and behaviors of growth cones leading to development of axon branches. We used bath and local application of a target-derived growth factor, FGF-2, on embryonic pyramidal neurons from the sensorimotor cortex and used time-lapse digital imaging to monitor effects of FGF-2 on axon branching. Observations of developing neurons over periods of several days showed that bath-applied FGF-2 significantly increased growth cone size and slowed growth cone advance, leading to a threefold increase in axon branching. FGF-2 also had acute effects on growth cone morphology, promoting rapid growth of filopodia within minutes. Application of FGF-2-coated beads promoted local axon branching in close proximity to the beads. Branching was more likely to occur when the FGF-2 bead was on or near the growth cone, suggesting that distal regions of the axon are more responsive to FGF-2 than other regions of the axon shaft. Together, these results show that interstitial axon branches can be induced locally through the action of a target-derived growth factor that preferentially exerts effects on the growth cone. We suggest that, in target regions, growth factors such as FGF-2 and other branching factors may induce formation of collateral axon branches by enhancing the pausing and enlargement of primary growth cones that determine future branch points.

Reorganization and movement of microtubules in axonal growth cones and developing interstitial branches.

Local changes in microtubule organization and distribution are required for the axon to grow and navigate appropriately; however, little is known about how microtubules (MTs) reorganize during directed axon outgrowth. We have used time-lapse digital imaging of developing cortical neurons microinjected with fluorescently labeled tubulin to follow the movements of individual MTs in two regions of the axon where directed growth occurs: the terminal growth cone and the developing interstitial branch. In both regions, transitions from quiescent to growth states were accompanied by reorganization of MTs from looped or bundled arrays to dispersed arrays and fragmentation of long MTs into short MTs. We also found that long-term redistribution of MTs accompanied the withdrawal of some axonal processes and the growth and stabilization of others. Individual MTs moved independently in both anterograde and retrograde directions to explore developing processes. Their velocities were inversely proportional to their lengths. Our results demonstrate directly that MTs move within axonal growth cones and developing interstitial branches. Our findings also provide the first direct evidence that similar reorganization and movement of individual MTs occur in the two regions of the axon where directed outgrowth occurs. These results suggest a model whereby short exploratory MTs could direct axonal growth cones and interstitial branches toward appropriate locations.

Altered GAP-43 immunoreactivity in regenerating sciatic nerve of diabetic rats.

Experimental diabetes in the rat is associated with impaired axon regeneration. Successful regeneration depends on the construction of axonal growth cones and establishment of appropriate target connections. The growth-associated protein (GAP)-43 is a major component of the axonal growth cone, and its synthesis and axonal transport are markedly increased during regeneration. The purpose of this study was to determine the effect of experimental diabetes on the synthesis and axonal transport of GAP-43 in regenerating sciatic nerves. Rats were rendered diabetic with 50 mg/kg streptozotocin i.p. Four weeks later, the rats were anesthetized, and one sciatic nerve was crushed to induce regeneration. After 2 weeks, nerves were ligated, and 6 h later, nerve pieces proximal to the ligature and dorsal root ganglia were removed, and proteins were separated by PAGE. Western blots of gels were probed with antibody 10E8/E7 against GAP-43. The presence of GAP-43 was confirmed by immunohistochemistry of nerve sections. Densitometric analysis of the blots showed a 45% reduction in native GAP-43 immunoreactivity in nerve pieces proximal to the ligature (P < 0.05; n = 7). Northern blots of total RNA extracted from pooled dorsal root ganglia were probed with a 32P-radiolabeled cDNA probe for GAP-43. There was no significant difference in the amount of GAP-43 mRNA between diabetic and nondiabetic rats. Immunohistochemistry of sciatic nerve confirmed the reduction in GAP-43 immunoreactivity. We conclude that a defect in turnover or axonal transport of GAP-43 may contribute to the impaired peripheral nerve regeneration in diabetes.