Predicting Cumulative and Maximum Brain Strain Measures From HybridIII Head Kinematics: A Combined Laboratory Study and <Emphasis Type="Italic">Post-Hoc</Emphasis> Regression Analysis

Due to growing concern on brain injury in sport, and the role that helmets could play in preventing brain injury caused by impact, biomechanics researchers and helmet certification organizations are discussing how helmet assessment methods might change to assess helmets based on impact parameters relevant to brain injury. To understand the relationship between kinematic measures and brain strain, we completed hundreds of impacts using a 50th percentile Hybrid III head-neck wearing an ice hockey helmet and input three-dimensional impact kinematics to a finite element brain model called the Simulated Injury Monitor (SIMon) (n = 267). Impacts to the helmet front, back and side included impact speeds from 1.2 to 5.8 ms^?1. Linear regression models, compared through multiple regression techniques, calculating adjusted R ² and the F-statistic, determined the most efficient set of kinematics capable of predicting SIMon-computed brain strain, including the cumulative strain damage measure (specifically CSDM-15) and maximum principal strain (MPS). Resultant change in angular velocity, Δω _R, better predicted CSDM-15 and MPS than the current helmet certification metric, peak g, and was the most efficient model for predicting strain, regardless of impact location. In nearly all cases, the best two-variable model included peak resultant angular acceleration, α _R, and Δω _R.     

Performance Evaluation of a Pre-computed Brain Response Atlas in Dummy Head Impacts

A pre-computed brain response atlas (pcBRA) may have the potential to accelerate the investigation of the biomechanical mechanisms of traumatic brain injury on a large-scale. In this study, we further enhance the technique and evaluate its performance using six degree-of-freedom angular velocity profiles from dummy head impacts. Using the pcBRA to simplify profiles into acceleration-only shapes, sufficiently accurate strain estimates were obtained for impacts with a major dominating velocity peak. However, they were largely under-estimated when substantial deceleration occurred that reversed the direction of the angular velocity. For these impacts, estimation accuracy was substantially improved with a biphasic profile simplification (average correlation coefficient and linear regression slope of 0.92 ± 0.03 and 0.95 ± 0.07 for biphasic shapes, respectively, vs. 0.80 ± 0.06 and 0.80 ± 0.08 for acceleration-only shapes). Peak maximum principal strain (? _p) and cumulative strain damage measure (CSDM) from the estimated strains consistently correlated stronger than kinematic metrics with respect to the baseline ? _p and CSDM from the directly simulated responses, regardless of the brain region, and by a large margin (e.g., correlation of 0.93 vs. 0.75 compared to Brain Injury Criterion (BrIC) for ? _p in the whole-brain, and 0.91 vs. 0.47 compared to BrIC for CSDM in the corpus callosum). These findings further support the pre-computation technique for accurate, real-time strain estimation, which could be important to accelerate model-based brain injury studies in the future.     

Individual Impact Magnitude vs. Cumulative Magnitude for Estimating Concussion Odds

Helmeted impact devices have allowed researchers to investigate the biomechanics of head impacts in vivo. While increased impact magnitude has been associated with greater concussion risk, a definitive concussive threshold has not been established. It is likely that concussion risk is not determined by a single impact itself, but a host of predisposing factors. These factors may include genetics, fatigue, and/or prior head impact exposure. The objective of the current paper is to investigate the association between cumulative head impact magnitude and concussion risk. It is hypothesized that increased cumulative magnitudes will be associated with greater concussion risk. This retrospective analysis included participants that were recruited from regional high-schools in Illinois and Michigan from 2007 to 2014 as part of an ongoing study on concussion biomechanics. Across seven seasons, 185 high school football athletes were instrumented with the Head Impact Telemetry system. Out of 185 athletes, 31 (17%) sustained a concussion, with two athletes sustaining two concussions over the study period, yielding 33 concussive events. The system recorded 78,204 impacts for all concussed players. Linear acceleration, rotational acceleration, and head impact telemetry severity profile (HITsp) magnitudes were summed within five timeframes: the day of injury, three days prior to injury, seven days prior to injury, 30 days prior to injury, and prior in-season exposure. Logistic regressions were modeled to explain concussive events based on the singular linear acceleration, rotational acceleration, and HITsp event along with the calculated summations over time. Linear acceleration, rotational acceleration, and HITsp all produced significant models estimating concussion (p < 0.05). The strongest estimators of a concussive impact were the linear acceleration (OR = 1.040, p < 0.05), rotational acceleration (OR = 1.001, p < 0.05), and HITsp (OR = 1.003, p < 0.05) for the singular impact rather than any of the cumulative magnitude calculations. Moreover, no cumulative count measure was significant for linear or rotational acceleration. Results from this investigation support the growing literature indicating cumulative magnitude is not related to concussion likelihood. Cumulative magnitude is a simplistic measure of the total exposure sustained by a player over a given period. However, this measure is limited as it assumes the brain is a static structure unable to undergo self-repair. Future research should consider how biological recovery between impacts may influence concussion risk.     

Development of an algorithm to automatically compress a CT image to visually lossless threshold

Background

To develop an algorithm to predict the visually lossless thresholds (VLTs) of CT images solely using the original images by exploiting the image features and DICOM header information for JPEG2000 compression and to evaluate the algorithm in comparison with pre-existing image fidelity metrics.

Methods

Five radiologists independently determined the VLT for 206 body CT images for JPEG2000 compression using QUEST procedure. The images were divided into training (n?=?103) and testing (n?=?103) sets. Using the training set, a multiple linear regression (MLR) model was constructed regarding the image features and DICOM header information as independent variables and regarding the VLTs determined with median value of the radiologists’ responses (VLT_rad) as dependent variable, after determining an optimal subset of independent variables by backward stepwise selection in a cross-validation scheme.The performance was evaluated on the testing set by measuring absolute differences and intra-class correlation (ICC) coefficient between the VLT_rad and the VLTs predicted by the model (VLT_model). The performance of the model was also compared two metrics, peak signal-to-noise ratio (PSNR) and high-dynamic range visual difference predictor (HDRVDP). The time for computing VLTs between MLR model, PSNR, and HDRVDP were compared using the repeated ANOVA with a post-hoc analysis. P?<?0.05 was considered to indicate a statistically significant difference.

Results

The means of absolute differences with the VLT_rad were 0.58 (95% CI, 0.48, 0.67), 0.73 (0.61, 0.85), and 0.68 (0.58, 0.79), for the MLR model, PSNR, and HDRVDP, respectively, showing significant difference between them (p?<?0.01). The ICC coefficients of MLR model, PSNR, and HDRVDP were 0.88 (95% CI, 0.81, 0.95), 0.85 (0.79, 0.91), and 0.84 (0.77, 0.91). The computing times for calculating VLT per image were 1.5?±?0.1 s, 3.9?±?0.3 s, and 68.2?±?1.4 s, for MLR metric, PSNR, and HDRVDP, respectively.

Conclusions

The proposed MLR model directly predicting the VLT of a given CT image showed competitive performance to those of image fidelity metrics with less computational expenses. The model would be promising to be used for adaptive compression of CT images.

     

Trunk Hybrid Passive–Active Musculoskeletal Modeling to Determine the Detailed T12–S1 Response Under <Emphasis Type="Italic">In Vivo</Emphasis> Loads

Biomechanical models of the spine either simplify intervertebral joints (using spherical joints or deformable beams) in musculoskeletal (MS) or overlook musculature in geometrically-detailed passive finite element (FE) models. These distinct active and passive models therefore fail to determine in vivo stresses and strains within and load-sharing among the joint structures (discs, ligaments, and facets). A novel hybrid active–passive spine model is therefore developed in which estimated trunk muscle forces from a MS model for in vivo activities drive a mechanically-equivalent passive FE model to quantify in vivo T12–S1 compression/shear loads, intradiscal pressures (IDP), centers of rotation (CoR), ligament/facet forces, and annulus fiber strains. The predicted and in vivo L4–L5 IDP and L1–S1 CoRs showed satisfactory agreements. The FE model under commonly-used in vitro loading (pure moments and follower loads) predicted different kinetics from those of the hybrid model under in vivo loads (muscle exertions and gravity loads) contributing to suggest the inadequacy of such in vitro loads when simulating in vivo tasks. For an improved assessment of the injury risk, evaluation of the internal loads, and design of implants, such hybrid models should therefore be used.     

Resting Heart Rate Variability (HRV) in Adolescents and Young Adults from a Genetically-Informed Perspective

Reduced heart rate variability (HRV) is associated with cardiac morbidity, mortality, and negative psychopathology. Most research concerning genetic influences on HRV has focused on adult populations, with fewer studies investigating the developmental period of adolescence and emerging adulthood. The current study estimated the genetic and environmental contributions to resting HRV in a sample of twins using various HRV time domain metrics to assess autonomic function across two different time measurement intervals (2.5- and 10-min). Five metrics of resting HRV [mean interbeat interval (IBI), the standard deviation of normal IBIs (SDNN), root square mean of successive differences between IBIs (RMSSD), cardiac vagal index (CVI), and cardiac sympathetic index (CSI)] were assessed in 421 twin pairs aged 14–20 during a baseline electrocardiogram. This was done for four successive 2.5-min intervals as well as the overall 10-min interval. Heritability (h²) appeared consistent across intervals within each metric with the following estimates (collapsed across time intervals): mean IBI (h²?=?0.36–0.46), SDNN (h²?=?0.23–0.30), RMSSD (h²?=?0.36–0.39), CVI (h²?=?0.37–0.42), CSI (h²?=?0.33–0.46). Beyond additive genetic contributions, unique environment also was an important influence on HRV. Within each metric, a multivariate Cholesky decomposition further revealed evidence of genetic stability across the four successive 2.5-min intervals. The same models showed evidence for both genetic and environmental stability with some environmental attenuation and innovation. All measures of HRV were moderately heritable across time, with further analyses revealing consistent patterns of genetic and environmental influences over time. This study confirms that in an adolescent sample, the time interval used (2.5- vs. 10-min) to measure HRV time domain metrics does not affect the relative proportions of genetic and environmental influences.     

γδT cells but not αβT cells contribute to sepsis-induced white matter injury and motor abnormalities in mice

Background

Infection and sepsis are associated with brain white matter injury in preterm infants and the subsequent development of cerebral palsy.

Methods

In the present study, we used a neonatal mouse sepsis-induced white matter injury model to determine the contribution of different T cell subsets (αβT cells and γδT cells) to white matter injury and consequent behavioral changes. C57BL/6J wild-type (WT), T cell receptor (TCR) δ-deficient (Tcrd ^?/?, lacking γδT cells), and TCRα-deficient (Tcra ^?/?, lacking αβT cells) mice were administered with lipopolysaccharide (LPS) at postnatal day (PND) 2. Brain myelination was examined at PNDs 12, 26, and 60. Motor function and anxiety-like behavior were evaluated at PND 26 or 30 using DigiGait analysis and an elevated plus maze.

Results

White matter development was normal in Tcrd ^?/? and Tcrα ^?/? compared to WT mice. LPS exposure induced reductions in white matter tissue volume in WT and Tcrα ^?/? mice, but not in the Tcrd ^?/? mice, compared with the saline-treated groups. Neither LPS administration nor the T cell deficiency affected anxiety behavior in these mice as determined with the elevated plus maze. DigiGait analysis revealed motor function deficiency after LPS-induced sepsis in both WT and Tcrα ^?/? mice, but no such effect was observed in Tcrd ^?/? mice.

Conclusions

Our results suggest that γδT cells but not αβT cells contribute to sepsis-induced white matter injury and subsequent motor function abnormalities in early life. Modulating the activity of γδT cells in the early stages of preterm white matter injury might represent a novel therapeutic strategy for the treatment of perinatal brain injury.

     

Prevalence of antiseptic resistance genes increases in staphylococcal isolates from orthokeratology lens wearers over initial six-month period of use

The purpose of this study was to investigate the prevalence of antiseptic-resistance (QAC) genes in staphylococci colonizing periorbital tissues and accessories of orthokeratology (ortho-k) lens wearers over a 6-month period and determine the effect of their presence on minimum inhibitory (MIC) and bactericidal concentrations (MBC) of disinfectants and log reduction of multipurpose contact lens solutions (MPS). Staphylococci were isolated from periorbital tissues and accessories of patients before commencing ortho-k therapy and at two subsequent visits. Presence of QAC genes in 116 S. aureus and 67 CNS isolates was determined by PCR and association with period of ortho-k use determined. MICs and MBCs of staphylococci gene-positive were compared with gene-negative strains and the effectiveness of four MPS for rigid contact lenses investigated. S. aureus carriage rates in the conjunctiva decreased significantly from 41.2 % (baseline) to 11.8 % (3-months), and 13.3 % (6-months) (p trend 0.03), while CNS increased from 58.8 %(baseline) to 94.1 % (3-months), and 93.3 % (6-months) (p trend 0.02). Prevalence of qacA/B increased considerably over time (S. aureus: 4.4 % to 15.4 %, CNS: 6.7 % to 25 %), but frequency of smr was relatively stable. Only five CNS isolates harboured qacH. MICs and MBCs of gene-positive isolates were significantly increased and three MPS did not achieve a 3-log reduction of many QAC-positive strains. Ortho-k lens wear contributed to changes of staphylococcal carriage rates in the conjunctival sac. Use of MPS containing quaternary ammonium compounds may select for the carriage of organisms harbouring QAC genes, as the low concentration of disinfecting agents is not adequate for killing gene-positive strains.     

Age is not associated with intracranial haemorrhage in patients with mild traumatic brain injury and oral anticoagulation

Background

Patients admitted to emergency departments with traumatic brain injury (TBI) are commonly being treated with oral anticoagulants. In contrast to patients without anticoagulant medication, no guidelines, scores or recommendations exist for the management of mild traumatic brain injury in these patients. We therefore tested whether age as one of the high risk factors of the Canadian head CT rule is applicable to a patient population on oral anticoagulants.

Methods

This cross-sectional analysis included all patients with mild TBI and concomitant oral anticoagulant therapy admitted to the Emergency Department, Inselspital Bern, Switzerland, from November 2009 to October 2014 (n?=?200). Using a logistic regression model, two groups of patients with mild TBI on oral anticoagulant therapy were compared — those with and those without intracranial haemorrhage.

Results

There was no significant difference in age between the patient groups with (n?=?86) and without (n?=?114) intracranial haemorrhage (p?=?0.078).In univariate logistic regression, GCS (OR?=?0.419 (0.258; 0.680)) and thromboembolic event as reason for anticoagulant therapy (OR?=?0.486 (0.257; 0.918)) were significantly associated with intracranial haemorrhage in patients with mild TBI and anticoagulation (all p?<?0.05). However, there was no association with age (p?=?0.078, OR?=?1.024 (0.997; 1.051)), the type of accident or additional medication with acetylsalicylic acid or clopidogrel ((both p?>?0.05; 0.552 (0.139; 2.202) and 0.256 (0.029; 2.237), respectively).

Conclusion

Our study found no association between age and intracranial bleeding. Therefore, until further risk factors are identified, diagnostic imaging with CCT remains necessary for mild TBI patients on oral anticoagulation of all ages, especially those with therapeutic anticoagulation because of thromboembolic events.

     

Magnetohydrodynamic Voltage Recorder for Comparing Peripheral Blood Flow

Blood flow is a clinical metric for monitoring of cardiovascular diseases but current measurements methods are costly or uncomfortable for patients. It was shown that the interaction of the magnetic field (B ₀) during MRI and blood flow in the body, through the magnetohydrodynamic (MHD) effect, produce voltages (V _MHD) observable through intra-MRI electrocardiography (ECG), which are correlated with regional blood flow. This study shows the reproducibility of V _MHD outside the MRI and its application in a portable flow monitoring device. To recreate this interaction outside the MRI, a static neodymium magnet (0.4T) was placed in between two electrodes to induce the V _MHD in a single lead ECG measurement. V _MHD was extracted, and integrated over to obtain a stroke volume metric. A smartphone-enabled device utilizing this interaction was developed in order to create a more accessible method of obtaining blood flow measurements. The portable device displayed a <6% error compared to a commercial recorder, and was able to successfully record V _MHD using the 0.4T magnet. Exercise stress testing showed a V _MHD increase of 23% in healthy subjects, with an 81% increase in the athlete. The study demonstrates a new device utilizing MHD interactions with body circulation to obtain blood flow metrics.     

Preparation of <Emphasis Type="Italic">Bordetella pertussis</Emphasis> DNA from respiratory samples for real-time PCR by commercial kits

Bordetella pertussis is increasingly detected by real-time PCR, but most kits for extracting Bordetella-DNA from respiratory samples are not validated for this material. Respiratory clinical materials were spiked with Bordetella pertussis cells. Four ion-exchange chromatography methods from one manufacturer were used for DNA preparation. Two real-time PCRs detecting the IS481 of Bordetella pertussis and based either on a hybridisation probes format (LightCycler®) or on a TaqMan® format were used. All kits effectively prepared DNA for Bordetella pertussis real-time PCR. The procedures were linear over a broad range, and the lower level of sensitivity was similar. Sensitivities measured as CT values were different. Inter-assay CVs were between 6.8% and 17.3%. Two kits did not effectively remove inhibitory substances from the respiratory samples. Commercial kits are useful for preparing Bordetella pertussis DNA from respiratory samples, but even kits from one manufacturer show significant differences in effectiveness and removal of inhibitory substances.     

EEG indices correlate with sustained attention performance in patients affected by diffuse axonal injury

The aim of this study is to assess the ability of EEG-based indices in providing relevant information about cognitive engagement level during the execution of a clinical sustained attention (SA) test in healthy volunteers and DAI (diffused axonal injury)-affected patients. We computed three continuous power-based engagement indices (P _β /P _α , 1/P _α , and P _β / (P _α + P _θ )) from EEG recordings in a control group (n = 7) and seven DAI-affected patients executing a 10-min Conners’ “not-X” continuous performance test (CPT). A correlation analysis was performed in order to investigate the existence of relations between the EEG metrics and behavioral parameters in both the populations. P _β /P _α and 1/P _α indices were found to be correlated with reaction times in both groups while P _β / (P _α + P _θ ) and P _β /P _α also correlated with the errors rate for DAI patients. In line with previous studies, time course fluctuations revealed a first strong decrease of attention after 2 min from the beginning of the test and a final fading at the end. Our results provide evidence that EEG-derived indices extraction and evaluation during SA tasks are helpful in the assessment of attention level in healthy subjects and DAI patients, offering motivations for including EEG monitoring in cognitive rehabilitation practice.

Open image in new window

Graphical abstract Three EEG-derived indices were computed from four electrodes montages in a population of seven healthy volunteers and a group of seven DAI-affected patients. Results show a significant correlation between the time course of the indices and behavioral parameters, thus demonstrating their usefulness in monitoring mental engagement level during a sustained attention task.

     

Comprehensive molecular biomarker identification in breast cancer brain metastases

Background

Breast cancer brain metastases (BCBM) develop in about 20–30% of breast cancer (BC) patients. BCBM are associated with dismal prognosis not at least due to lack of valuable molecular therapeutic targets. The aim of the study was to identify new molecular biomarkers and targets in BCBM by using complementary state-of-the-art techniques.

Methods

We compared array expression profiles of three BCBM with 16 non-brain metastatic BC and 16 primary brain tumors (prBT) using a false discovery rate (FDR) p < 0.05 and fold change (FC) > 2. Biofunctional analysis was conducted on the differentially expressed probe sets. High-density arrays were employed to detect copy number variations (CNVs) and whole exome sequencing (WES) with paired-end reads of 150 bp was utilized to detect gene mutations in the three BCBM.

Results

The top 370 probe sets that were differentially expressed between BCBM and both BC and prBT were in the majority comparably overexpressed in BCBM and included, e.g. the coding genes BCL3, BNIP3, BNIP3P1, BRIP1, CASP14, CDC25A, DMBT1, IDH2, E2F1, MYCN, RAD51, RAD54L, and VDR. A number of small nucleolar RNAs (snoRNAs) were comparably overexpressed in BCBM and included SNORA1, SNORA2A, SNORA9, SNORA10, SNORA22, SNORA24, SNORA30, SNORA37, SNORA38, SNORA52, SNORA71A, SNORA71B, SNORA71C, SNORD13P2, SNORD15A, SNORD34, SNORD35A, SNORD41, SNORD53, and SCARNA22. The top canonical pathway was entitled, role of BRCA1 in DNA damage response. Network analysis revealed key nodes as Akt, ERK1/2, NFkB, and Ras in a predicted activation stage. Downregulated genes in a data set that was shared between BCBM and prBT comprised, e.g. BC cell line invasion markers JUN, MMP3, TFF1, and HAS2. Important cancer genes affected by CNVs included TP53, BRCA1, BRCA2, ERBB2, IDH1, and IDH2. WES detected numerous mutations, some of which affecting BC associated genes as CDH1, HEPACAM, and LOXHD1.

Conclusions

Using complementary molecular genetic techniques, this study identified shared and unshared molecular events in three highly aberrant BCBM emphasizing the challenge to detect new molecular biomarkers and targets with translational implications. Among new findings with the capacity to gain clinical relevance is the detection of overexpressed snoRNAs known to regulate some critical cellular functions as ribosome biogenesis.

     

Development of an <Emphasis Type="Italic">In Vitro</Emphasis> 3D Brain Tissue Model Mimicking <Emphasis Type="Italic">In Vivo</Emphasis>-Like Pro-inflammatory and Pro-oxidative Responses

To analyze complex inflammatory responses in an in vitro system, we constructed a new 3D in vitro brain tissue model that exhibits in vivo-like tissue responses (e.g. immune cell phenotypes, and molecular response) to inflammatory stimuli. Finite element modeling of oxygen diffusion and cellular oxygen consumption predicted the oxygen profile within 3D structures, consisting of Type I collagen hydrogel embedded with murine microglia. Viability and cytotoxicity analyses supported the mathematical analysis, determining optimal cell growth conditions for 3D construct development. Real-time RT-PCR and ELISA demonstrated significant up-regulation of pro-inflammatory mediators, such as TNF-α, MCP-1, IL-6 and IL-1β, in lipopolysaccharide (LPS)-stimulated in vitro cell culture (2D and 3D) and in vivo mouse model systems. Interestingly, levels of inflammatory responses from the in vitro 3D model system were more similar to in vivo than in vitro 2D. Additionally, in situ dihydroethidium (DHE) assay and immunofluorescence staining revealed that levels of LPS-stimulated reactive oxygen species (ROS) generation and microglial activation from in vitro 3D model system were closer to in vivo than in vitro 2D. These results demonstrated that an in vitro 3D model provides more physiologically relevant pro-oxidative and pro-inflammatory environments in brain than an in vitro 2D model.     

Effect of lipopolysaccharide on circadian clock genes <Emphasis Type="Italic">Per2</Emphasis> and <Emphasis Type="Italic">Bmal1</Emphasis> in mouse ovary

In mammals, circadian rhythms are associated with multiple physiological events. The aim of the present study was to examine the effect of lipopolysaccharide (LPS) on circadian systems in the ovary. Immature female mice were received an intra-peritoneal injection of equine chorionic gonadotropin (eCG) and LPS. Total RNA was collected from the ovary at 6-h intervals throughout a 48 h of experimental period. The expression of the circadian genes period 2 (Per2) and brain and muscle ARNT-like 1 (Bmal1) such as circadian genes was measured by quantitative PCR. Although expression of Per2 and Bmal1 in the ovary did not display clear diurnal oscillation, LPS suppressed the amplitude of Per2 expression. Additionally, LPS inhibited the expression of cytochrome P450 aromatase (CYP19) and luteinizing hormone receptor (LHr) genes in the ovary of eCG-treated mice. Our data suggest that Per2 may be associated with the inhibition of CYP19 and LHr expression by LPS in the ovaries of immature mice.     

Ectoparasites of dogs and cats in Albania

One hundred eighty-one dogs and 26 short-hair cats from suburban areas around Tirana, Albania were examined for ectoparasite infestation. The dogs were examined on several occasions from 2005 through 2009 representing three seasons: winter (December–February), spring (March–May), and summer (June–August); the cats were examined in late autumn (November). In addition, deep ear swab specimens of 30 dogs were examined for ear mites. The arthropod ectoparasite fauna of the dogs included two tick species (Rhipicephalus sanguineus and Ixodes ricinus), three mite species (Sarcoptes scabiei var. canis, Otodectes cynotis, and Demodex canis), three flea species (Ctenocephalides canis, Ctenocephalides felis, and Pulex irritans), and one louse species (Trichodectes canis). In the dogs, rates of infestation were 23.8% for R. sanguineus, 0.6% for I. ricinus, 4.4% for S. scabiei var. canis, 6.7% for O. cynotis, 0.6% for D. canis, 75.7% for C. canis, 5.0% for C. felis, 8.3% for P. irritans, and 6.6% for T. canis. Mixed infestation with two or three species of ectoparasites was recorded on 38.1% of the dogs. Fleas infested 75.7% dogs (geometric mean, 3.96; range, 1–80) and were observed in winter, spring, and summer with increasing prevalences of 64.3%, 75.9%, and 100%. Ticks parasitized 24.3% of the dogs (geometric mean, 0.41; range, 1–331). R. sanguineus ticks were recorded on 34.2% and 50% of the dogs examined in spring and summer, respectively, but were absent on the dogs during winter except for a single I. ricinus specimen observed. Prevalence of infestation with R. sanguineus, S. scabiei var. canis, C. felis, P. irritans, and T. canis did not differ between dogs ≤6 months and dogs >6 months of age; however, prevalence of infestation with C. canis was significantly (p?<?0.01) higher in dogs >6 months old. There was no difference between the sexes for the prevalences of infestation with those parasites. The examination of the cats revealed infestation with only one species of ectoparasite, C. felis (prevalence, 100%; geometric mean, 2.5; range, 1–9).     

Major components of the insulin-like growth factor axis are expressed early in chicken embryogenesis,with IGF binding protein (<Emphasis Type="Italic">IGFBP</Emphasis>)<Emphasis Type="Italic">-5</Emphasis> expression subject to regulation by Sonic Hedgehog

Using whole-mount in situ hybridisation techniques, we have examined the expression of major components of the insulin-like growth factor (IGF) axis in early development of the chicken embryo, including both IGF-I and -II, the type 1 IGF receptor (IGFR), and two of the IGF binding proteins, (IGFBP)-2 and -5. We report that these genes fall into two distinct groups with respect to expression pattern, with IGFBP-2 displaying broad overlap of mRNA expression with IGFR and IGF-I during early development, whereas the expression profile of IGFBP-5 most closely resembled that of IGF-II. Comparison between different stages revealed IGFBP-2 mRNA was detected as early as stage 3, whereas IGFBP-5 was first seen at stage 4. In addition, we detected expression domains of IGFBP-5, and to a lesser extent IGFBP-2, which did not overlap with either IGFR or IGF expression patterns. This could indicate IGF independent actions of the IGFBPs during early embryonic development. A striking observation concerning the expression profiles of both IGF-II and IGFBP-5 at early stages of chick embryogenesis is that both these genes are expressed asymmetrically in a pattern similar to that of Sonic Hedgehog (Shh). Furthermore, using cyclopamine, we have demonstrated that IGFBP-5 expression in the early embryo is regulated by Shh. Taken together, these results describe an important role for the IGF system in the very early stages of the developing chicken embryo, and imply that IGFBP-2 and -5 are fundamental developmental factors, with the latter involved in Shh signalling pathways.