A Diagnostic Test for the Mixing Distribution in a Generalised Linear Mixed Model

We introduce a diagnostic test for the mixing distribution in a generalised linear mixed model. The test is based on the difference between the marginal maximum likelihood and conditional maximum likelihood estimates of a subset of the fixed effects in the model. We derive the asymptotic variance of this difference, and propose a test statistic that has a limiting chi-square distribution under the null hypothesis that the mixing distribution is correctly specified. For the important special case of the logistic regression model with random intercepts, we evaluate via simulation the power of the test in finite samples under several alternative distributional forms for the mixing distribution. We illustrate the method by applying it to data from a clinical trial investigating the effects of hormonal contraceptives in women.

Experimental model to evaluate in vivo and in vitro cartilage MR imaging by means of histological analyses

OBJECTIVES: Implementation of an experimental model to compare cartilage MR imaging by means of histological analyses. MATERIAL AND METHODS: MRI was obtained from 4 patients expecting total knee replacement at 1.5 and/or 3T prior surgery. The timeframe between pre-op MRI and knee replacement was within two days. Resected cartilage-bone samples were tagged with Ethi((R))-pins to reproduce the histological cutting course. Pre-operative scanning at 1.5T included following parameters for fast low angle shot (FLASH: TR/TE/FA=33ms/6ms/30 degrees , BW=110kHz, 120mmx120mm FOV, 256x256 matrix, 0.65mm slice-thickness) and double echo steady state (DESS: TR/TE/FA=23.7ms/6.9ms/40 degrees , BW=130kHz, 120x120mm FOV, 256x256 matrix, 0.65mm slice-thickness). At 3T, scan parameters were: FLASH (TR/TE/FA=12.2ms/5.1ms/10 degrees , BW=130kHz, 170x170mm FOV, 320x320, 0.5mm slice-thickness) and DESS (TR/TE/FA=15.6ms/4.5ms/25 degrees , BW=200kHz, 135mmx150mm FOV, 288x320matrix, 0.5mm slice-thickness). Imaging of the specimens was done the same day at 1.5T. MRI (Noyes) and histological (Mankin) score scales were correlated using the paired t-test. Sensitivity and specificity for the detection of different grades of cartilage degeneration were assessed. Inter-reader and intra-reader reliability was determined using Kappa analysis. RESULTS: Low correlation (sensitivity, specificity) was found for both sequences in normal to mild Mankin grades. Only moderate to severe changes were diagnosed with higher significance and specificity. The use of higher field-strengths was advantageous for both protocols with sensitivity values ranging from 13.6% to 93.3% (FLASH) and 20.5% to 96.2% (DESS). Kappa values ranged from 0.488 to 0.944. CONCLUSIONS: Correlating MR images with continuous histological slices was feasible by using three-dimensional imaging, multi-planar-reformat and marker pins. The capability of diagnosing early cartilage changes with high accuracy could not be proven for both FLASH and DESS.

Granulocyte colony stimulating factor supports liver regeneration in a surgical small for size liver remnant mouse model

ntense liver regeneration and almost 100% survival follows partial hepatectomy of up to 70% of liver mass in rodents. More extensive resections of 70 to 80% have an increased mortality and partial hepatectomies of >80% constantly lead to acute hepatic failure and death in mice. The aim of the study was to determine the effect of systemically administered granulocyte colony stimulating factor (G-CSF) on animal survival and liver regeneration in a small for size liver remnant mouse model after 83% partial hepatectomy (liver weight <0.8% of mouse body weight). Methods: Male Balb C mice (n=80, 20-24g) were preconditioned daily for five days with 5μg G-CSF subcutaneously or sham injected (aqua ad inj). Subsequently 83% hepatic resection was performed and daily sham or G-CSF injection continued. Survival was determined in both groups (G-CSF n=35; Sham: n=33). In a second series BrdU was injected (50mg/kg Body weight) two hours prior to tissue harvest and animals euthanized 36 and 48 hours after 83% liver resection (n=3 each group). To measure hepatic regeneration the BrdU labeling index and Ki67 expression were determined by immunohistochemistry by two independent observers. Harvested liver tissue was dried to constant weight at 65 deg C for 48 hours. Results: Survival was 0% in the sham group on day 3 postoperatively and significantly better (26.2% on day 7 and thereafter) in the G-CSF group (Log rank test: p<0.0001). Dry liver weight was increased in the G-CSF group (T-test: p<0.05) 36 hours after 83% partial hepatectomy. Ki67 expression was elevated in the G-CSF group at 36 hours (2.8±2.6% (Standard deviation) vs 0.03±0.2%; Rank sum test: p<0.0001) and at 48 hours (45.1±34.6% vs 0.7±1.0%; Rank sum test: p<0.0001) after 83% liver resection. BrdU labeling at 48 hours was 0.1±0.3% in the sham and 35.2±34.2% in the G-CSF group (Rank sum test: p<0.0001) Conclusions: The surgical 83% resection mouse model is suitable to test hepatic supportive regimens in the setting of small for size liver remnants. Administration of G-CSF supports hepatic regeneration after microsurgical 83% partial hepatectomy and leads to improved long-term survival in the mouse. G-CSF might prove to be a clinically valuable supportive substance in small for size liver remnants in humans after major hepatic resections due to primary or secondary liver tumors or in the setting of living related liver donation.

Call for a reference model of chronic hind limb ischemia to investigate therapeutic angiogenesis

A large number of studies utilize animal models to investigate therapeutic angiogenesis. However, the lack of a standardized experimental model leaves the comparison of different studies problematic. To establish a reference model of prolonged moderate tissue ischemia, we created unilateral hind limb ischemia in athymic rnu-rats by surgical excision of the femoral vessels. Blood flow of the limb was monitored for 60 days by laser Doppler imaging. Following a short postoperative period of substantially depressed perfusion, the animals showed a status of moderate hind limb ischemia from day 14 onwards. Thereafter, the perfusion remained at a constant level (55.5% of normal value) until the end of the observation period. Histopathological assessment of the ischemic musculature on postoperative days 28 and 60 showed essentially no inflammatory cell infiltrate or fibrosis. However, the mitochondrial activity and capillary-to-fiber ratio of the muscular tissue was reduced to 52.7% of normal, presenting with a significant weakness of the ischemic limb evidenced by a progressive decline in performance. Intramuscular injection of culture-expanded human endothelial progenitor cells (EPC) resulted in a significant increase in blood flow (82.0+/-3.5% of normal), capillary density (1.60+/-0.08/muscle fiber) and smooth muscle covered arterioles (8.0+/-0.6/high power field) in the ischemic hind limb as compared to controls (55.0+/-3.1%; 0.99+/-0.03; 5.0+/-0.2). In conclusion, chronic, moderate hind limb ischemia with consistently reduced perfusion levels persisting over a prolonged period can be established reliably in rnu athymic nude rats and is responsive to pro-angiogenic treatments such as EPC transplantation. This study provides a detailed protocol of a highly reproducible reference model to test novel therapeutic options for limb ischemia.

Complex visual hallucinations and attentional performance in eye disease and dementia: a test of the Perception and Attention Deficit model

OBJECTIVE This study aimed to test the prediction from the Perception and Attention Deficit model of complex visual hallucinations (CVH) that impairments in visual attention and perception are key risk factors for complex hallucinations in eye disease and dementia. METHODS Two studies ran concurrently to investigate the relationship between CVH and impairments in perception (picture naming using the Graded Naming Test) and attention (Stroop task plus a novel Imagery task). The studies were in two populations-older patients with dementia (n = 28) and older people with eye disease (n = 50) with a shared control group (n = 37). The same methodology was used in both studies, and the North East Visual Hallucinations Inventory was used to identify CVH. RESULTS A reliable relationship was found for older patients with dementia between impaired perceptual and attentional performance and CVH. A reliable relationship was not found in the population of people with eye disease. CONCLUSIONS The results add to previous research that object perception and attentional deficits are associated with CVH in dementia, but that risk factors for CVH in eye disease are inconsistent, suggesting that dynamic rather than static impairments in attentional processes may be key in this population.

Potentially avoidable 30-day hospital readmissions in medical patients: derivation and validation of a prediction model

IMPORTANCE Because effective interventions to reduce hospital readmissions are often expensive to implement, a score to predict potentially avoidable readmissions may help target the patients most likely to benefit. OBJECTIVE To derive and internally validate a prediction model for potentially avoidable 30-day hospital readmissions in medical patients using administrative and clinical data readily available prior to discharge. DESIGN Retrospective cohort study. SETTING Academic medical center in Boston, Massachusetts. PARTICIPANTS All patient discharges from any medical services between July 1, 2009, and June 30, 2010. MAIN OUTCOME MEASURES Potentially avoidable 30-day readmissions to 3 hospitals of the Partners HealthCare network were identified using a validated computerized algorithm based on administrative data (SQLape). A simple score was developed using multivariable logistic regression, with two-thirds of the sample randomly selected as the derivation cohort and one-third as the validation cohort. RESULTS Among 10 731 eligible discharges, 2398 discharges (22.3%) were followed by a 30-day readmission, of which 879 (8.5% of all discharges) were identified as potentially avoidable. The prediction score identified 7 independent factors, referred to as the HOSPITAL score: h emoglobin at discharge, discharge from an o ncology service, s odium level at discharge, p rocedure during the index admission, i ndex t ype of admission, number of a dmissions during the last 12 months, and l ength of stay. In the validation set, 26.7% of the patients were classified as high risk, with an estimated potentially avoidable readmission risk of 18.0% (observed, 18.2%). The HOSPITAL score had fair discriminatory power (C statistic, 0.71) and had good calibration. CONCLUSIONS AND RELEVANCE This simple prediction model identifies before discharge the risk of potentially avoidable 30-day readmission in medical patients. This score has potential to easily identify patients who may need more intensive transitional care interventions.

Last interglacial temperature evolution — a model inter-comparison

There is a growing number of proxy-based reconstructions detailing the climatic changes that occurred during the last interglacial period (LIG). This period is of special interest, because large parts of the globe were characterized by a warmer-than-present-day climate, making this period an interesting test bed for climate models in light of projected global warming. However, mainly because synchronizing the different palaeoclimatic records is difficult, there is no consensus on a global picture of LIG temperature changes. Here we present the first model inter-comparison of transient simulations covering the LIG period. By comparing the different simulations, we aim at investigating the common signal in the LIG temperature evolution, investigating the main driving forces behind it and at listing the climate feedbacks which cause the most apparent inter-model differences. The model inter-comparison shows a robust Northern Hemisphere July temperature evolution characterized by a maximum between 130–125 ka BP with temperatures 0.3 to 5.3 K above present day. A Southern Hemisphere July temperature maximum, −1.3 to 2.5 K at around 128 ka BP, is only found when changes in the greenhouse gas concentrations are included. The robustness of simulated January temperatures is large in the Southern Hemisphere and the mid-latitudes of the Northern Hemisphere. For these regions maximum January temperature anomalies of respectively −1 to 1.2 K and −0.8 to 2.1 K are simulated for the period after 121 ka BP. In both hemispheres these temperature maxima are in line with the maximum in local summer insolation. In a number of specific regions, a common temperature evolution is not found amongst the models. We show that this is related to feedbacks within the climate system which largely determine the simulated LIG temperature evolution in these regions. Firstly, in the Arctic region, changes in the summer sea-ice cover control the evolution of LIG winter temperatures. Secondly, for the Atlantic region, the Southern Ocean and the North Pacific, possible changes in the characteristics of the Atlantic meridional overturning circulation are crucial. Thirdly, the presence of remnant continental ice from the preceding glacial has shown to be important when determining the timing of maximum LIG warmth in the Northern Hemisphere. Finally, the results reveal that changes in the monsoon regime exert a strong control on the evolution of LIG temperatures over parts of Africa and India. By listing these inter-model differences, we provide a starting point for future proxy-data studies and the sensitivity experiments needed to constrain the climate simulations and to further enhance our understanding of the temperature evolution of the LIG period.

Do different anesthesia regimes affect hippocampal apoptosis and neurologic deficits in a rodent cardiac arrest model?

Background Different anesthesia regimes are commonly used in experimental models of cardiac arrest, but the effects of various anesthetics on clinical outcome parameters are unknown. We conducted a study in which we subjected rats to cardiac arrest under medetomidine/ketamine or sevoflurane/fentanyl anesthesia. Methods Asystolic cardiac arrest for 8 minutes was induced in 73 rats with a mixture of potassium chloride and esmolol. Daily behavioral and neurological examination included the open field test (OFT), the tape removal test (TRT) and a neurodeficit score (NDS). Animals were randomized for sacrifice on day 2 or day 5 and brains were harvested for histology in the hippocampus cornus ammonis segment CA1. The inflammatory markers IL-6, TNF-α, MCP-1 and MIP-1α were assessed in cerebrospinal fluid (CSF). Proportions of survival were tested with the Fisher’s exact test, repeated measurements were assessed with the Friedman’s test; the baseline values were tested using Mann–Whitney U test and the difference of results of repeated measures were compared. Results In 31 animals that survived beyond 24 hours neither OFT, TRT nor NDS differed between the groups; histology was similar on day 2. On day 5, significantly more apoptosis in the CA1 segment of the hippocampus was found in the sevoflurane/fentanyl group. MCP-1 was higher on day 5 in the sevoflurane/fentanyl group (p = 0.04). All other cyto- and chemokines were below detection threshold. Conclusion In our cardiac arrest model neurological function was not influenced by different anesthetic regimes; in contrast, anesthesia with sevoflurane/fentanyl results in increased CSF inflammation and histologic damage at day 5 post cardiac arrest.

Use of activity monitors for assessment of pruritus in an acute model of canine atopic dermatitis

BACKGROUND: We developed a canine model of acute atopic dermatitis to evaluate the potential of compounds to treat pruritus and skin lesions induced in Dermatophagoides farinae (Df)-sensitized dogs. HYPOTHESIS/OBJECTIVES: The aim was to investigate the effectiveness of long-term recording activity monitors to assess pruritus induced by allergen challenges. ANIMALS: Thirty-two Df-sensitized laboratory dogs. METHODS: In two blinded crossover studies, 28 Df-sensitized dogs were challenged on 3 days with a Df slurry applied to clipped abdominal skin. Dogs were treated with a positive control (prednisolone 1 mg/kg once daily for 5 days, starting 1 day before challenge) or left untreated; all were fitted with activity monitors. To confirm pruritus, a parallel study with four dogs was conducted, filming the dogs before and during challenge and assessing the film for pruritic behaviour. RESULTS: The activity of dogs treated with prednisolone was significantly lower between 00.00 and 03.00 h and between 03.00 and 06.00 h compared with untreated dogs (repeated-measures ANCOVA; P < 0.0001). To determine whether the recorded night-time activity corresponded to pruritic manifestations, we compared activity monitor and video recordings of four dogs for two periods (16.30-20.30 and 24.00-03.00 h) before and during a Df challenge. The correlation between night-time activity monitor activity and observed pruritic behaviour was highly significant (test of correlation coefficient versus zero: r = 0.57, P < 0.0001). CONCLUSIONS AND CLINICAL IMPORTANCE: Determination of night-time activity with activity monitors after allergen challenge appears to be an objective and practical way to assess pruritus in this experimental model of canine atopic dermatitis.

An improved simple rat model for global cerebral ischaemia by induced cardiac arrest

OBJECTIVES Cerebral hypoxic-ischaemic injury following cardiac arrest is a devastating disease affecting thousands of patients each year. There is a complex interaction between post-resuscitation injury after whole-body ischaemia-reperfusion and cerebral damage which cannot be explored in in vitro systems only; there is a need for animal models. In this study, we describe and evaluate the feasibility and efficiency of our simple rodent cardiac arrest model. METHODS Ten wistar rats were subjected to 9 and 10 minutes of cardiac arrest. Cardiac arrest was introduced with a mixture of the short-acting beta-blocking drug esmolol and potassium chloride. RESULTS All animals could be resuscitated within 1 minute, and survived until day 5.General health score and neurobehavioural testing indicated substantial impairment after cardiac arrest, without differences between groups. Histological examination of the hippocampus CA1 segment, the most vulnerable segment of the cerebrum, demonstrated extensive damage in the cresyl violet staining, as well as in the Fluoro-Jade B staining and in the Iba-1 staining, indicating recruitment of microglia after the hypoxic-ischaemic event. Again, there were no differences between the 9- and 10-minute cardiac arrest groups. DISCUSSION We were able to establish a simple and reproducible 9- and 10-minute rodent cardiac arrest models with a well-defined no-flow-time. Extensive damage can be found in the hippocampus CA1 segment. The lack of difference between 9- and 10-minute cardiac arrest time in the neuropsychological, the open field test and the histological evaluations is mainly due to the small sample size.

A randomized trial of the effects of the noble gases helium and argon on neuroprotection in a rodent cardiac arrest model.

BACKGROUND The noble gas xenon is considered as a neuroprotective agent, but availability of the gas is limited. Studies on neuroprotection with the abundant noble gases helium and argon demonstrated mixed results, and data regarding neuroprotection after cardiac arrest are scant. We tested the hypothesis that administration of 50% helium or 50% argon for 24 h after resuscitation from cardiac arrest improves clinical and histological outcome in our 8 min rat cardiac arrest model. METHODS Forty animals had cardiac arrest induced with intravenous potassium/esmolol and were randomized to post-resuscitation ventilation with either helium/oxygen, argon/oxygen or air/oxygen for 24 h. Eight additional animals without cardiac arrest served as reference, these animals were not randomized and not included into the statistical analysis. Primary outcome was assessment of neuronal damage in histology of the region I of hippocampus proper (CA1) from those animals surviving until day 5. Secondary outcome was evaluation of neurobehavior by daily testing of a Neurodeficit Score (NDS), the Tape Removal Test (TRT), a simple vertical pole test (VPT) and the Open Field Test (OFT). Because of the non-parametric distribution of the data, the histological assessments were compared with the Kruskal-Wallis test. Treatment effect in repeated measured assessments was estimated with a linear regression with clustered robust standard errors (SE), where normality is less important. RESULTS Twenty-nine out of 40 rats survived until day 5 with significant initial deficits in neurobehavioral, but rapid improvement within all groups randomized to cardiac arrest. There were no statistical significant differences between groups neither in the histological nor in neurobehavioral assessment. CONCLUSIONS The replacement of air with either helium or argon in a 50:50 air/oxygen mixture for 24 h did not improve histological or clinical outcome in rats subjected to 8 min of cardiac arrest.

Adaptation and maladaptation of heart and skeletal muscle to different diets in a rodent model of human obesity

Obesity and diabetes are metabolic disorders associated with fatty acid availability in excess of the tissues' capacity for fatty acid oxidation. This mismatch is implicated in the pathogenesis of cardiac contractile dysfunction and also in skeletal muscle insulin resistance. My dissertation will present work to test the overall hypothesis that "western" and high fat diets differentially affect cardiac and skeletal muscle fatty acid oxidation, the expression of fatty acid responsive genes, and cardiac contractile function. Wistar rats were fed a low fat, "western," or high fat (10%, 45%, or 60% calories from fat, respectively) diet for acute (1 day to 1 week), short (4 to 8 weeks), intermediate (16 to 24 weeks), or long (32 to 48 weeks) term. With high fat diet, cardiac oleate oxidation increased at all time points investigated. In contrast, with western diet cardiac oleate oxidation increased in the acute, short and intermediate term, but not in the long term. Consistent with a maladaptation of fatty acid oxidation, cardiac power (measured ex vivo) decreased with long term western diet only. In contrast to the heart, soleus muscle oleate oxidation increased only in the acute and short term with either western or high fat feeding. Transcript analysis revealed that several fatty acid responsive genes, including pyruvate dehydrogenase kinase 4, uncoupling protein 3, mitochondrial thioesterase 1, and cytosolic thioesterase 1 increased in heart and soleus muscle to a greater extent with high fat diet, versus western diet, feeding. In conclusion, the data implicate inadequate induction of a cassette of fatty acid responsive genes in both the heart and skeletal muscle by western diet resulting in impaired activation of fatty acid oxidation, and the development of cardiac dysfunction. ^

RESPIRATORY COMPENSATION MECHANISMS IN THE UPPER AND LOWER RESPIRATORY TRACTS OF AN ANIMAL MODEL AFTER SUBCHRONIC INHALATION EXPOSURE TO FORMALDEHYDE: IMPLICATIONS FOR TOXICOLOGY RISK ASSESSMENT (DEPOSITION, DOSE RECEIVED, RESPONSE)

The potential for significant human populations to experience long-term inhalation of formaldehyde and reports of symptomatology due to this exposure has led to a considerable interest in the toxicologic assessment of risk from subchronic formaldehyde exposures using animal models. Since formaldehyde inhalation depresses certain respiratory parameters in addition to its other forms of toxicity, there is a potential for the alteration of the actual dose received by the exposed individual (and the resulting toxicity) due to this respiratory effect. The respiratory responses to formaldehyde inhalation and the subsequent pattern of deposition were therefore investigated in animals that had received subchronic exposure to the compound, and the potential for changes in the formaldehyde dose received due to long-term inhalation evaluated. Male Sprague-Dawley rats were exposed to either 0, 0.5, 3, or 15 ppm formaldehyde for 6 hours/day, 5 days/week for up to 6 months. The patterns of respiratory response, deposition and the compensation mechanisms involved were then determined in a series of formaldehyde test challenges to both the upper and to the lower respiratory tracts in separate groups of subchronically exposed animals and age-specific controls (four concentration groups, two time points). In both the control and pre-exposed animals, there was a characteristic recovery of respiratory parameters initially depressed by formaldehyde inhalation to at or approaching pre-exposure levels within 10 minutes of the initiation of exposure. Also, formaldehyde deposition was found to remain very high in the upper and lower tracts after long-term exposure. Therefore, there was probably little subsequent effect on the dose received by the exposed individual that was attributable to the repeated exposures. There was a diminished initial minute volume response in test challenges of both the upper and lower tracts of animals that had received at least 16 weeks of exposure to 15 ppm, with compensatory increases in tidal volume in the upper tract and respiratory rate in the lower tract. However, this dose-related effect was probably not relevant to human risk estimation because this formaldehyde dose is in excess of that experienced by human populations. ^