Dihydropyrimidinase related protein-2 as a biomarker for temperature and time dependent post mortem changes in the mouse brain proteome

Proteome analysis in the central nervous system area represents a large and important challenge in drug discovery. One major problem is to obtain representative and well characterized tissues of high quality for analysis. We have used brain tissues from normal mice to study the effect of post mortem time (up to 32 h) and temperature (4 degrees C and room temperature) on protein expression patterns. A number of proteins were identified using mass spectrometry and potential markers were localized. One of the proteins identified, dihydropyrimidinase related protein-2 (DRP-2), occurs as multiple spots in two-dimensional electrophoresis gels. The ratio between the truncated form of DRP-2 (fDRP-2) and full length DRP-2 is suggested as an internal control that can be used as a biomarker of post mortem time and post mortem temperature between unrelated brain protein samples. Results of this study may be useful in future efforts to detect disease specific alterations in proteomic studies of human post mortem brain tissues.     

Survival of Trichomonas gallinae in white-winged dove carcasses

Survival of Trichomonas gallinae was examined in white-winged dove (Zenaida asiatica) carcasses to assess whether birds that have been dead up to 8 hr can be sampled reliably for this protozoan. Carcasses of 100 T. gallinae-positive white-winged doves were separated into four groups of 25 birds, representing 2, 4, 6, and 8 hr post mortem sampling intervals and placed into an environmental chamber maintained at 27 C and 75% relative humidity. Live T. gallinae were isolated in 96, 100, 100, and 92% of the carcasses at each of the respective post mortem intervals. The experiment was repeated with another 100 carcasses of T. gallinae-positive white-winged doves placed in the environmental chamber, this time maintained at 27 C and 40% relative humidity. Live T. gallinae occurred in 96, 100, 96, and 100% of the carcasses at each of the respective post mortem intervals. Across both trials, the overall ability to detect positive birds from sampling carcasses up to 8 hrs post mortem was 97%. An a posteriori experiment was conducted in which 23 and 18 carcasses from the second trial were maintained in the environmental chamber at 27 C and 40% relative humidity and resampled at 24 and 48 hr post mortem, respectively. Live trichomonads were isolated from 91 and 44% of the carcasses at 24 and 48 hr, respectively. Results suggest live T. gallinae can be obtained from dove carcasses reliably up to 8 hr and possibly up to 24 hr after host death. The ability for T. gallinae to survive within this time interval can aid wildlife personnel in monitoring this protozoan at hunter check stations or obtaining samples from recently killed birds.     

First investigations to refine video-based IR thermography as a non-invasive tool to monitor the body temperature of calves

In this study, a video-based infrared camera (IRC) was investigated as a tool to monitor the body temperature of calves. Body surface temperatures were measured contactless using videos from an IRC fixed at a certain location in the calf feeder. The body surface temperatures were analysed retrospectively at three larger areas: the head area (in front of the forehead), the body area (behind forehead) and the area of the entire animal. The rectal temperature served as a reference temperature and was measured with a digital thermometer at the corresponding time point. A total of nine calves (Holstein-Friesians, 8 to 35 weeks old) were examined. The average maximum temperatures of the area of the entire animal (mean±SD: 37.66±0.90°C) and the head area (37.64±0.86°C) were always higher than that of the body area (36.75±1.06°C). The temperatures of the head area and of the entire animal were very similar. However, the maximum temperatures as measured using IRC increased with an increase in calf rectal temperature. The maximum temperatures of each video picture for the entire visible body area of the calves appeared to be sufficient to measure the superficial body temperature. The advantage of the video-based IRC over conventional IR single-picture cameras is that more than one picture per animal can be analysed in a short period of time. This technique provides more data for analysis. Thus, this system shows potential as an indicator for continuous temperature measurements in calves.     

Post-mortem glycolysis in ox skeletal muscle. Effect of temperature on the concentrations of glycolytic intermediates and cofactors

1. Post-mortem changes in the concentrations of the following compounds in ox sternomandibularis muscles stored in nitrogen at 1 degrees , 5 degrees and 15 degrees are reported: P(i) creatine phosphate, hexose monophosphates, fructose diphosphate, triose phosphates, alpha-glycerophosphate, phosphoglycerates, lactate, ATP, ADP, AMP, NAD(+) and total nucleotides. Some results obtained with muscles stored at 37 degrees are included. 2. At the time the muscles were placed at controlled temperatures (about 1.5hr. post mortem) the phosphorus in the compounds measured accounted for 91+/-6% (s.d.) of the total acid-soluble phosphate. 3. The results indicated that at all temperatures the activities of the phosphorylase and phosphofructokinase steps limited the rate and the extent of post-mortem glycolysis. 4. The large variations in hexose monophosphate concentrations during storage indicated that the ratio of phosphorylase to phosphofructokinase activity varied considerably with time and temperature. 5. Between 3.5 and 7hr. post mortem the rates of glycolysis and of ATP turnover were not slower at 5 degrees that at 15 degrees , and were probably faster at 1 degrees . The significance of this finding is discussed.     

Systematic investigation of degradation effects on spin-spin relaxation times in mouse-liver by low resolution NMR

Despite numerous work on spin-lattice (T1) relaxation in vitro, not much attention has been paid on spin-spin (T2) relaxation until now. In this study we are presenting spin-spin relaxation time measurements of mouse liver tissue in order to estimate the time-after-excision effects. The post mortem behaviour of excised tissue was investigated up to four hours in intervals of about nine minutes. The time course of liver T2 was determined for different temperatures (4 degrees - 40 degrees C) for female mice. In order to describe the similar behaviour of T2 and pH changes in mouse liver after excision, we are suggesting an empirical model to correlate this data. In contrast to T1 results published recently, we found no significant differences in liver T2 time course after excision due to different physiological states like sex, starvation or circadian rhythm. T1/T2-behaviour after tissue excision is discussed in an attempt to separate various relaxation mechanisms.     

Post mortem changes in skeletal muscle proton spin-lattice relaxation times

A previously published report indicated that there are early post mortem changes in the pulsed NMR proton spin-lattice relaxation time (T1) of skeletal muscle which must be taken into account in in vitro tissue analysis. We re-examined this subject. When T1 measurements were done by allowing the signal intensity to decay over two orders of magnitude from the original intensity, the T1 decay curves showed more than a single exponential relaxation time component as reported earlier. However, when T1 measurements were done by allowing signal intensity to decay to about one order of magnitude from the original intensity only a single exponential relaxation time component was found. We postulate that the latter method gives the average T1 value of the various macroscopic tissue components present and that this method gives a representative T1 value for the entire tissue. Using data obtained in this manner we could not find significant post mortem changes in the muscle T1 relaxation times during the first four hours but found change at a later time.     

Comparative studies on lipogenic enzyme activities in the liver of human and some animal species

1. The activities of enzymes involved in fatty acid synthesis in the human liver (sample taken during abdominal surgery) and in the livers of some animals were studied. 2. Fatty acid synthase, ATP-citrate lyase and malic enzyme activities were found to be from 4 to 70-fold lower in human liver than in rat or bird livers. 3. The activities of hexose monophosphate shunt dehydrogenases in human liver were from half to almost equal to the corresponding activities in birds, but much lower than in rat liver. 4. The activities of all enzymes listed above in human and beef liver were very similar (except fatty acid synthase which was undetectable in the beef liver). 5. Very high activity of NADP-linked isocitrate dehydrogenase was found in livers of all species tested. 6. These results are discussed in relation to the role of the human liver in lipogenesis. 7. The activities of the enzymes generating NADPH in human liver taken during abdominal surgery were similar to the activities observed in the tissue obtained post mortem. 8. This suggested that post mortem tissue may be used as a reliable human material for some enzyme assays. 9. Thus we also examined the activity of malic enzyme in post mortem human kidney cortex, heart, skeletal muscle and brain. 10. Relatively high activity of NADP-linked malic enzyme has been observed in human brain.     

Comparison of porcine brain mechanical properties to potential tissue simulant materials in quasi-static and sinusoidal compression

In both finite element and physical surrogate models of head blast injury, accurate material properties of the brain and/or tissue simulants are necessary to ensure biofidelity in predicted response. Thus, there is a need for experimental comparisons between tissue and simulant materials under the same experimental conditions. This study compares the response of porcine brain tissue and a variety of brain tissue simulants in quasi-static and sinusoidal compression tests. Fresh porcine brain tissue was obtained from a local abattoir and tested within 4 h post mortem. Additionally, the effect of post mortem time was investigated by comparing samples stored at room temperature and stored frozen (−18 °C), at various time intervals. The brain tissue simulants tested were bovine gelatin (3%, 5%, and 10% concentration), agarose gelatin (e0.4%, 0.6%, 0.8% concentration), and Sylgard 527. The experiments were performed using a DMA apparatus (TA Instruments Q800). The quasi-static compression data were fit to Ogden hyperelastic functions so that parameters could be compared. It was found that bovine gelatin at 3% and 5% concentration demonstrated the closest response to brain tissue in quasi-static compression. Conversely, in sinusoidal compression, the agarose gel and Sylgard 527 were found to be in closer agreement with the tissue, than bovine gel. In terms of post mortem time and storage, there was no statistically significant difference detected in the response of tissue samples after 48 h, regardless of storage method. However, samples stored at room temperature after 48 h appeared to demonstrate a reduction in stiffness.     

Thermophysiological consequences of whole body resonant RF exposure (100 MHz) in human volunteers

Thermophysiological responses of heat production and heat loss were measured in seven adult volunteers (six males and one female, aged 31-74 years) during 45 min dorsal exposures of the whole body to 100 MHz continuous wave (CW) radio frequency (RF) energy. Three power densities (PD) (average PD = 4, 6, and 8 mW/cm(2); whole body specific absorption rate [SAR] = 0.068 [W/kg]/[mW/cm(2)]) were tested in each of three ambient temperatures (T(a) = 24, 28, and 31 degrees C), as well as in T(a) controls (no RF). A standardized protocol (30 min baseline, 45 min RF or sham exposure, 10 min baseline) was used. Measured responses included esophageal and seven skin temperatures, metabolic heat production, local sweat rate, and local skin blood flow. No changes in metabolic heat production occurred under any test condition. Unlike published results of similar exposures at 450 and 2450 MHz, local skin temperatures, even those on the back that were irradiated directly, changed little or not at all during 100 MHz exposures. The sole exception was the temperature of the ankle skin, which increased by 3-4 degrees C in some subjects at PD = 8 mW/cm(2). During the 45 min RF exposure, esophageal temperature showed modest changes (range = -0.15 to 0.13 degrees C) and never exceeded 37.2 degrees C. Thermoregulation was principally controlled by appropriate increases in evaporative heat loss (sweating) and, to a lesser extent, by changes in skin blood flow. Because of the deep penetration of RF energy at this frequency, effectively bypassing the skin, these changes must have been stimulated by thermal receptors deep in the body rather than those located in the skin.     

Effect of time post mortem on the concentration of endotoxin in rat organs: implications for sudden infant death syndrome (SIDS)

The aim of the study was to test the following hypotheses: (i) that endotoxin injected 40 min prior to death can be detected in rat organs post mortem and (ii) that endotoxin levels do not change with increasing time post mortem. Rats were injected with or without endotoxin in buffered saline, 40 min prior to being killed. Endotoxin levels in rat organs were assessed using a Limulus amoebocyte assay. The effect of storage time post mortem was assessed by following various storage regimes at 25 degrees C and 8 degrees C. Significant differences (P = < 0.001) in endotoxin levels of all samples tested were found between rats injected with and without endotoxin. A significant increase in detectable endotoxin was observed between 0 h and 6 h post mortem in rats injected with or without endotoxin. No difference in detectable endotoxin levels in the kidney, liver and spleen was observed from 30 h to 102 h post mortem in rats injected with or without endotoxin. In rats injected with endotoxin, detectable endotoxin levels in the heart were raised between 0 h and 6 h, 6 h and 54 h, and 30 h and 78 h. Endotoxin injected into rats 40 min prior to death can be detected post mortem. For rats injected with saline or endotoxin prior to death levels in the kidney, liver and spleen were not affected by storage at 8 degrees C for 30-102 h, after initial storage at room temperature for 6 h. Levels of endotoxin detected in the hearts of rats injected with saline were not affected by storage up to 102 h. In rats injected with endotoxin prior to death, detectable levels in the heart were significantly affected by increasing time in storage.     

Microwave irradiation and brain γ-aminobutyric acid levels in mice

Microwave irradiation sufficient to raise the core brain temperatures of mice to >60°C is essential to prevent post mortem increases in γ-aminobutyric acid levels. Physiological or drug-induced changes in the body temperatures of mice profoundly effect the final temperatures of the brains after microwave treatment and may lead to spurious values for putative neurotransmitter concentration estimates. This paper points out the importance of certain data each laboratory $\text{must}$ establish for itself when applying microwave technique.     

Effect of delay in cryofixation on the elemental composition of biopsies and post mortem specimens of the thyroid gland

The time between the excision and cryofixation of a biopsy is most important regarding its elemental composition as demostrated by an investigation of the thyroid glands of rats and pigs. Biopsies taken and cryofixed immediately served as control specimens. Biopsies that were allowed to stand at room temperature for 20 min before cryofixation and specimens cryofixed at 1 h post mortem were also investigated. Significant changes in the ion concentration of the cells and colloid were apparent in biopsies in which cryofixation was delayed for 20 min and in thyroids cryofixed 1 h post mortem. It was demonstrated that redistribution of electrolytes occurs within 1h post mortem and that similar changes occur in biopsies allowed to stand for 20 min at room temperature before cryofixation. The results stress the importance of immediate cryofixation after surgical excision of a biopsy. This is especially important since numerous elemental changes due to delayed cryofixation resemble those which occur in pathological processes.     

Acoustic thermometry of the patient brain with traumatic brain injury

Non-invasive deep brain acoustic thermometry is carried out for two patients at Burdenko Neurosurgery Institute. This method is based on the measurements of the own thermal acoustic radiation of the investigated object. These two patients have got the brain injury. Some of their skull bones are absent. Infrared thermometry was also used to measure the surface temperature of the forehead skin. On the basis of the experimental data the temperatures deep within the brain were reconstructed. The values for the two patients are equal to 37.3 ± 0.7 and 37.0 ± 0.3°C.     

Systematic investigation of degradation effects on spin-lattice relaxation times in mouse-liver by low resolution NMR

In spite of numerous work on in vitro proton relaxation time investigations of biological tissue, many questions still remain open. In this study we focused on spin-lattice (T1) relaxation time measurements of mouse liver tissue in order to estimate the time-after-excision effects. The post mortem behaviour of excised tissue was measured up to four hours in intervals of about nine minutes. The time course of liver T1 was determined for different temperatures (4 degrees-40 degrees C) for female mice and the effect of starvation (up to 48 hours) on the time course of T1 was investigated for male and female mice at 37 degrees C. We obtained significant differences in liver T1 time course after excision due to different physiological states like sex, starvation and circadian rhythm.     

Correlation of in vivo testicle and seminal vesicle size with post mortem dimensions in bulls

This study was conducted to quantify the relationships between in vivo measurements of testicular and seminal vesicle size and post mortem size of these organs in 30 Santa Gertrudis bulls. The in vivo measurements of testicles were obtained by transrectal ultrasonography and palpation per rectum, while scrotal circumference was measured by scrotal tape. Linear post mortem dimensions were obtained by direct measurements of the excised organs. Volume was assessed by water displacement while the testicles were weighed. Seminal vesicle length, determined by palpation, had the highest correlation with post mortem measurements (r = 0.70; P = 0.0001). Accurate estimation of the thickness of the vesicles (1.47 vs 1.55 cm for in vivo and post mortem, respectively) was performed by ultrasonograph. Of all seminal vesicle linear measurements, width had the highest correlations with volume measured by water displacement (r = 0.67; P = 0.0001 and r = 0.38; P = 0.04 for post mortem and in vivo, respectively). Testicular diameter was accurately measured by ultrasonography (5.54 vs 4.58 cm in vivo and post mortem, respectively) and was highly correlated (range r = 0.84 to 0.89; P = 0.0001) with post mortem measurements of testicular volume, weight and circumference. The correlation between scrotal circumference and diameter of the testicle was 0.75 (P = 0.0001). The correlations of testicular diameter measured by ultrasound with the post mortem measurements of testicular weight and circumference were similar to the correlations between scrotal circumference and those 2 post mortem measurements. We conclude that palpation of vesicle length is highly correlated with volume of the seminal vesicle in situ. Individual linear measurements do not seem to be an accurate predictor of the relativ size of the seminal vesicle. Furthermore, ultrasonography does not seem to be a more accurate measure of testicular size than scrotal circumference for evaluation of breeding soundness.     

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.     

The spin-lattice relaxation times of water associated with early post mortem changes in skeletal muscle.

We studied the spin-echo signal of muscle water in a large time domain and found that the motion of the nuclear magnetic moment of tissue water cannot be characterized by a single spin-lattice relaxation time (T1). The relaxation time T1B, which is the T1 characterized by those protons with a slower relaxation rate, is influenced by the early post mortem changes in skeletal muscle. T1B increased with time after the tissue was taken from the animal and reached a maximum at 3 h. However, the weighted average of T1 of all water protons (T1A) did not change throughout the time course of the experiments.     

Persistence of sleep-temperature coupling after suprachiasmatic nuclei lesions in rats

The suprachiasmatic nucleus (SCN) regulates the circadian rhythms of body temperature (T(b)) and vigilance states in mammals. We studied rats in which circadian rhythmicity was abolished after SCN lesions (SCNx rats) to investigate the association between the ultradian rhythms of sleep-wake states and brain temperature (T(br)), which are exposed after lesions. Ultradian rhythms of T(br) (mean period: 3.6 h) and sleep were closely associated in SCNx rats. Within each ultradian cycle, nonrapid eye movement (NREM) sleep was initiated 5 +/- 1 min after T(br) peaks, after which temperature continued a slow decline (0.02 +/- 0.006 degrees C/min) until it reached a minimum. Sleep and slow wave activity (SWA), an index of sleep intensity, were associated with declining temperature. Cross-correlation analysis revealed that the rhythm of T(br) preceded that of SWA by 2-10 min. We also investigated the thermoregulatory and sleep-wake responses of SCNx rats and controls to mild ambient cooling (18 degrees C) and warming (30 degrees C) over 24-h periods. SCNx rats and controls responded similarly to changes in ambient temperature. Cooling decreased REM sleep and increased wake. Warming increased T(br), blunted the amplitude of ultradian T(br) rhythms, and increased the number of transitions into NREM sleep. SCNx rats and controls had similar percentages of NREM sleep, REM sleep, and wake, as well as the same average T(b) within each 24-h period. Our results suggest that, in rats, the SCN modulates the timing but not the amount of sleep or the homeostatic control of sleep-wake states or T(b) during deviations in ambient temperature.     

Loss of EphA4 impairs short‐term spatial recognition memory performance and locomotor habituation

EphA4 receptor (EphA4) tyrosine kinase is an important regulator of central nervous system development and synaptic plasticity in the mature brain, but its relevance to the control of normal behavior remains largely unexplored. This study is the first attempt to obtain a behavioral profile of constitutive homozygous and heterozygous EphA4 knockout mice. A deficit in locomotor habituation in the open field, impairment in spatial recognition in the Y‐maze and reduced probability of spatial spontaneous alternation in the T‐maze were identified in homozygous EphA4^?/? mice, while heterozygo us EphA4^+/? mice appeared normal on these tests in comparison with wild‐type (WT) controls. The multiple phenotypes observed in EphA4^?/? mice might stem from an underlying deficit in habituation learning, reflecting an elementary form of nonassociative learning that is in contrast to Pavlovian associative learning, which appeared unaffected by EphA4 disruption. A deficit in motor coordination on the accelerating rotarod was also demonstrated only in EphA4^?/? mice – a finding in keeping with the presence of abnormal gait in EphA4^?/? mice – although they were able to improve performance over training. There was no evidence for substantial changes in major neurochemical markers in various brain regions rich in EphA4 as shown by post‐mortem analysis. This excludes the possibility of major neurochemical compensation in the brain of EphA4^?/? mice. In summary, we have demonstrated for the first time the behavioral significance of EphA4 disruption, supporting further investigation of EphA4 as a possible target for behavioral interventions where habituation deficits are prominent.