Continuous Monitoring of Post Mortem Temperature Changes in the Human Brain

Recent developments in neurochemistry research on the post mortem human brain require a detailed understanding of the post mortem changes in the human brain, including the correlation between time related temperature changes and alterations in biochemical parameters. As an initial step towards our deeper insight into the intricate relationships between post mortem time, temperature and neurochemical processes, in the present study we set out to monitor continuously temperature changes in the post mortem human brain in eight cadavers for a period of up to 24 h after death under 'standard' clinical conditions at a neurosurgery clinic. A main objective of the study was to find a simple and reliable mathematical formula, requiring only time and an easily obtainable body temperature measurement parameter, with the help of which the superficial and deep brain temperatures can be obtained without invasive interactions. With a portable thermoprobe data logger system superficial (4 cm from skull surface) and deep (8 cm) brain temperatures, the temperature of the liver and that of the forehead skin, as well as the ambient temperature of the room were measured at regular time intervals (every 1 or 5 min). Various mathematical models were fitted to the data in order to create a simple model capable to predict brain temperatures from easily accessible measurements, such as that of the forehead skin. On the basis of the tested models we propose that with simple polynomial equations the deep and superficial brain temperatures can be described reliably as T (br4) ( degrees C)=T (fh)-0.001t (3)+0.0541t (2)-1.0622t+7.5933 and T (br8) ( degrees C)=T (fh)-0.0003t (3)+0.0201t (2)-0.619t+7.9036, respectively, where T (br4) is the superficial (4 cm) brain temperature, T (br8) is the deep (8 cm) brain temperature, T (fh) is the forehead temperature and t is the time from death. These measurements can, in combination with further neurochemical studies, contribute to our better understanding of the human brain's time- and temperature-related post mortem biochemical changes.     

Cerebellar Oxidative DNA Damage and Altered DNA Methylation in the BTBR T+tf/J Mouse Model of Autism and Similarities with Human Post Mortem Cerebellum

The molecular pathogenesis of autism is complex and involves numerous genomic, epigenomic, proteomic, metabolic, and physiological alterations. Elucidating and understanding the molecular processes underlying the pathogenesis of autism is critical for effective clinical management and prevention of this disorder. The goal of this study is to investigate key molecular alterations postulated to play a role in autism and their role in the pathophysiology of autism. In this study we demonstrate that DNA isolated from the cerebellum of BTBR T+tf/J mice, a relevant mouse model of autism, and from human post-mortem cerebellum of individuals with autism, are both characterized by an increased levels of 8-oxo-7-hydrodeoxyguanosine (8-oxodG), 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC). The increase in 8-oxodG and 5mC content was associated with a markedly reduced expression of the 8-oxoguanine DNA-glycosylase 1 (Ogg1) and increased expression of de novo DNA methyltransferases 3a and 3b (Dnmt3a and Dnmt3b). Interestingly, a rise in the level of 5hmC occurred without changes in the expression of ten-eleven translocation expression 1 (Tet1) and Tet2 genes, but significantly correlated with the presence of 8-oxodG in DNA. This finding and similar elevation in 8-oxodG in cerebellum of individuals with autism and in the BTBR T+tf/J mouse model warrant future large-scale studies to specifically address the role of OGG1 alterations in pathogenesis of autism.     

The Use of Contrast-Enhanced Post Mortem CT in the Detection of Cardiovascular Deaths

Objectives

To evaluate the diagnostic value of contrast enhanced post mortem computed tomography (PMCT) in comparison to non-enhanced post mortem CT in the detection of cardiovascular causes of death (COD).

Background

As autopsy rates decline, new methods to determine CODs are necessary. So contrast enhanced PMCT shall be evaluated in comparison to established non-enhanced PMCT in order to further improve the method.

Methods

In a prospective study, 20 corpses were examined using a 64-row multisclice CT (MSCT) before and after intraarterial perfusion with a newly developed, barium-bearing contrast agent and ventilation of the lungs. The cause of death was determined in enhanced and unenhanced scans and a level of confidence (LOC) was given by three experienced radiologists on a scale between 0 and 4. Results were compared to autopsy results as gold standard. Autopsy was performed blinded to PMCT-findings.

Results

The method allowed visualization of different types of cause of death. There was a significant improvement in LOC in enhanced scans compared to unenhanced scans as well as an improvement in the detection of COD. The cause of death could be determined in 19 out of 20 patients.

Conclusions

PMCT is feasible and appears to be robust for diagnosing cardiovascular causes of death. When compared with unenhanced post-mortem CT intraarterial perfusion and pulmonary ventilation significantly improve visualization and diagnostic accuracy. These promising results warrant further studies.     

Comparison of Magnetic Resonance Imaging in Live vs. Post Mortem Rat Brains

Magnetic Resonance Imaging (MRI) is an increasingly popular technique for examining neurobiology in rodents because it is both noninvasive and nondestructive. MRI scans can be acquired from either live or post mortem specimens. In vivo scans have a key advantage in that subjects can be scanned at multiple time-points in longitudinal studies. However, repeated exposure to anesthesia and stress may confound studies. In contrast, post mortem scans offer improved image quality and increased signal-to-noise ratio (SNR) due to several key advantages: First, the images are not disrupted by motion and pulsation artifacts. Second, they allow the brain tissue to be perfused with contrast agents, enhancing tissue contrast. Third, they allow longer image acquisition times, yielding higher resolution and/or improved SNR. Fourth, they allow assessment of groups of animals at the same age without scheduling complications. Despite these advantages, researchers are often skeptical of post mortem MRI scans because of uncertainty about whether the fixation process alters the MRI measurements. To address these concerns, we present a thorough comparative study of in vivo and post mortem MRI scans in healthy male Wistar rats at three age points throughout adolescence (postnatal days 28 through 80). For each subject, an in vivo scan was acquired, followed by perfusion and two post mortem scans at two different MRI facilities. The goal was to assess robustness of measurements, to detect any changes in volumetric measurements after fixation, and to investigate any differential bias that may exist between image acquisition techniques. We present this volumetric analysis for comparison of 22 anatomical structures between in vivo and post mortem scans. No significant changes in volumetric measurements were detected; however, as hypothesized, the image quality is dramatically improved in post mortem scans. These findings illustrate the validity and utility of using post mortem scans in volumetric neurobiological studies.     

Causes of Stillbirth and Time of Death in Swedish Holstein Calves Examined Post Mortem

This study was initiated due to the observation of increasing and rather high levels of stillbirths, especially in first-calving Swedish Holstein cows (10.3%, 2002). Seventy-six Swedish Holstein calves born to heifers at 41 different farms were post mortem examined in order to investigate possible reasons for stillbirth and at what time in relation to full-term gestation they had occurred. The definition of a stillborn calf was dead at birth or within 24 h after birth after at least 260 days of gestation. Eight calves were considered as having died already in uterus. Slightly less than half of the examined calves (46.1%) were classified as having died due to a difficult calving. Four calves (5.3%) had different kinds of malformations (heart defects, enlarged thymus, urine bladder defect). Approximately one third of the calves (31.6%) were clinically normal at full-term with no signs of malformation and born with no indication of difficulties at parturition or any other reason that could explain the stillbirth. The numbers of male and female calves were rather equally distributed within the groups. A wide variation in post mortem weights was seen in all groups, although a number of the calves in the group of clinically normal calves with unexplained reason of death were rather small and, compared with e.g. those calves categorised as having died due to a difficult calving, their average birth weight was 6 kg lower (39.9 ± 1.7 kg vs. 45.9 ± 1.5 kg, p ≤ 0.01). It was concluded that the cause of stillbirth with a non-infectious aetiology is likely to be multifactorial and difficult calving may explain only about half of the stillbirths. As much as one third of the calves seemed clinically normal with no obvious reason for death. This is a target group of calves that warrants a more thorough investigation in further studies.     

Salt Stress Induced Nuclear and DNA Degradation in Meristematic Cells of Barley Roots

Root growth of barley (Hordeum vulgare L., cv. Akashinriki)was inhibited by 200 raM NaCl, when 1 mM CaCl₂ was present inthe hydroponic culture solution. Increasing the CaCl₂ up to10 mM partially prevented this inhibition. However, inhibitionalso occurred with 100 mM NaCl in the presence of 0.1 mM CaCl₂.The nuclei of meristematic cells in roots in which growth hadbeen inhibited by salt stress were studied after staining withDAPI (4',6-diamino-2-phenylindol). Nuclear deformation of thecells occurred with 12 h of salt stress with 500 mM NaCl, andwas followed by degradation. The nuclear degradation was alsoobserved when the roots were exposed to more than 300 mM NaClfor 24 h. Biochemical analysis revealed that nuclear degradationwas accompanied by apoptosis-like DNA fragmentation. The intracellularmechanisms of nuclear degradation in cells after salt stressare discussed. ¹Emertius professor, Okayama University.     

Comparative Proteomic Profiling of Divergent Phenotypes for Water Holding Capacity across the Post Mortem Ageing Period in Porcine Muscle Exudate

Two dimensional Difference Gel Electrophoresis (2-D DIGE) and mass spectrometry were applied to investigate the changes in metabolic proteins that occur over a seven day (day 1, 3 and 7) post mortem ageing period in porcine centrifugal exudate from divergent meat quality phenotypes. The objectives of the research were to enhance our understanding of the phenotype (water holding capacity) and search for biomarkers of this economically significant pork quality attribute. Major changes in protein abundance across nine phenotype-by-time conditions were observed. Proteomic patterns were dominated by post mortem ageing timepoint. Using a machine learning algorithm (l1-regularized logistic regression), a model was derived with the ability to discriminate between high drip and low drip phenotypes using a subset of 25 proteins with an accuracy of 63%. Models discriminating between divergent phenotypes with accuracy of 72% and 73% were also derived comparing respectively, high drip plus intermediate phenotype (considered as one phenotype) versus low drip and comparing low drip plus intermediate phenotype (considered as one phenotype) versus high drip. In all comparisons, the general classes of discriminatory proteins identified include metabolic enzymes, stress response, transport and structural proteins. In this research we have enhanced our understanding of the protein related processes underpinning this phenotype and provided strong data to work toward development of protein biomarkers for water holding capacity.     

Partial Genome Scale Analysis of Gene Expression in Human Adipose Tissue Using DNA Array

Objective: Large scale analysis of gene expression in adipose tissue provides a basis for the identification of novel candidate genes involved in the pathophysiology of obesity. Our goal was to explore gene expression in human adipose tissue at a partial genome scale using DNA array. Research Methods and Procedures: Labeled cDNA, derived from human adipose tissue poly(A⁺) RNA, was hybridized to a DNA array containing over 18,000 human expressed sequence‐tagged (EST) clones. The results were analyzed by database searches. Results: Homology searches of the 300 EST clones with highest hybridization signals revealed that 145 contained DNA sequences identical to known genes and 79 could be linked to UniGene clusters. Of the 145 identified genes, 136 were nonredundant and subsequently characterized with respect to function and chromosomal localization by searching MEDLINE, UniGene, GeneMap, OMIM, SWISS‐PROT, the Genome Database, and the Location Data Base. The identified genes were grouped according to their putative functions; cell/organism defense (9.6%), cell division (5.1%), cell signaling/communication (19.8%), cell structure/motility (12.5%), gene/protein expression (16.9%), metabolism (16.2%), and unclassified (19.8%). Less than 50% of these genes have previously been reported to be expressed in adipose tissue. The chromosomal localization of 268 genes strongly expressed in adipose tissue showed that their relative abundance was significantly increased on chromosomes 11, 19, and 22 compared to the expected distribution of the same number of random genes. Discussion: Our study resulted in the identification of numerous genes previously not reported to be expressed in adipose tissue. These results suggest that DNA array is a powerful tool in the search for novel regulatory pathways within adipose tissue on a scale that is not possible using conventional methods.     

Effect of Dietary Magnesium on Post Mortem Phosphocreatine Utilization in Skeletal Muscle of Swine: A Non-Invasive Study Using 31P-NMR Spectroscopy

The effect of dietary magnesium on the post mortem PCr (phosphocreatine) decay in muscle of heterozygote malignant hyperthermia pigs was studied after in vivo exposure to a combination of halothane and succinylcholine. The pigs were anaesthetized with halothane and succinylcholine was injected in the ear vein. Immediately after initiation of the depolarizing neuromuscular blocking effect of succinylcholine the animals were captive-bolt stunned. The PCr decay, reflecting ATP turnover, was followed in situ by 31P-NMR spectroscopy in the biceps femoris muscle for the subsequent 40-70 min post mortem. In 3 of the 4 experiments, the Mg-fed pig had a significantly reduced rate of PCr hydrolysis compared to the control animal. The mechanism of this magnesium effect is unknown.     

Correlation between Fibrillin-1 Degradation and mRNA Downregulation and Myofibroblast Differentiation in Cultured Human Dental Pulp Tissue

Myofibroblasts and extracellular matrix are important components in wound healing. Alpha-smooth muscle actin (α-SMA) is a marker of myofibroblasts. Fibrillin-1 is a major constituent of microfibrils and an extracellular-regulator of TGF-β1, an important cytokine in the transdifferentiation of resident fibroblasts into myofibroblasts. To study the correlation between changes in fibrillin-1 expression and myofibroblast differentiation, we examined alterations in fibrillin-1 and α-SMA expression in organotypic cultures of dental pulp in vitro. Extracted healthy human teeth were cut to 1-mm-thick slices and cultured for 7 days. In intact dental pulp, fibrillin-1 was broadly distributed, and α-SMA was observed in pericytes and vascular smooth muscle cells. After 7 days of culture, immunostaining for fibrillin-1 became faint concomitant with a downregulation in its mRNA levels. Furthermore, fibroblasts, odontoblasts and Schwann cells were immunoreactive for α-SMA with a significant increase in α-SMA mRNA expression. Double immunofluorescence staining was positive for pSmad2/3, central mediators of TGF-β signaling, and α-SMA. The administration of inhibitors for extracellular matrix proteases recovered fibrillin-1 immunostaining; moreover, fibroblasts lost their immunoreactivity for α-SMA along with a downregulation in α-SMA mRNA. These findings suggest that the expression of α-SMA is TGF-β1 dependent, and fibrillin-1 degradation and downregulation might be implicated in the differentiation of myofibroblasts in dental pulp wound healing.     

Effects of Dietary Energy Sources on Post Mortem Glycolysis,Meat Quality and Muscle Fibre Type Transformation of Finishing Pigs

Dietary energy source can influence muscle glycogen storage at slaughter. However, few studies have demonstrated whether the diet-induced change of muscle glycogen is achieved by the transformation of muscle fibre type. This study investigated the effects of dietary energy sources on meat quality, post mortem glycolysis and muscle fibre type transformation of finishing pigs. Seventy-two barrows with an average body weight of 65.0 ± 2.0 kg were selected and were allotted to three iso-energetic and iso-nitrogenous diets A, B or C, and each treatment consisted of three replicates (pens) of eight pigs each. Diet A contained 44.1% starch, 5.9% crude fat and 12.6% neutral detergent fiber (NDF); diet B contained 37.6% starch, 9.5% crude fat and 15.4% NDF; and diet C contained 30.9% starch, 14.3% crude fat and 17.8% NDF. The duration of the experiment was 28 days. After feed withdrawal 12 h, 24 pigs (eight per treatment) were slaughtered, samples from M. longissimus lumborum (LL) were collected for subsequent analysis. The results showed that pigs fed diet C had lesser average daily gain, average daily feed intake and back fat depth than those fed diet A (P<0.05). Diet C increased pH_45min (P<0.05) and decreased drip loss (P<0.05) in LL muscles compared with diet A. Meat from pigs fed diet A showed increased contents of lactate and greater glycolytic potential (GP) compared with those fed diet C (P<0.05). Greater mRNA expression of myosin heavy-chain (MyHC)-I and IIa and lesser expression of MyHC-IIx and IIb (P<0.05) in LL muscles were found in pigs fed diet C, than in pigs fed diet A. In addition, pigs fed diet C resulted in downregulation of miR23a and upregulation of miR409 and miR208b (P<0.05), associated with conserved changes of their corresponding targets. These findings indicated that diets containing low starch and high fibre were beneficial in reducing muscle glycolysis, improving meat quality of finishing pigs. This reduction of GP may be partially associated with the improvement of oxidative fibre composition in LL muscle, and the change in myofibre type may be correlated with the change in the miRNA expression.     

酶降解法精制肝素钠

肝素钠在哺乳动物的脏器和体液如：肝、肺、脾及胃肠粘膜等中含量很高．肝素钠作为一种有效的抗凝剂已广泛应用于临床,生物学功能在于阻止血液中凝血酶的作用．其衍生物还具有降血脂、消炎、抗过敏、利尿及调节免疫功能等多种用途．肝素钠属于多糖类化合物,作为溶栓类药...     

The Use of a Fabric Softener in the Reconstitution of Mummified Tissue Prior to Paraffin Wax Sectioning for Light Microscopical Examination

A method of reconstituting mummified tissues using the fabric softener “Comfort” is described. Routine histological paraffin wax sections were prepared following overnight treatment at room temperature with a 0.2% solution of this substance in isotonic saline. A comparison of results obtained by this method is made with those obtained using Ruffer's and Sandison's solutions. Nuclear stains gave poor results whereas connective tissue stains demonstrated their respective fibers by all rehydration methods.