A Comparison of Methodologies for the Study of Functional Transmitter Neurochemistry in Human Brain

A number of different approaches to the study of functional neurochemistry in human brain are discussed. The advantages and disadvantages of three main techniques are contrasted: (i) using animal tissue preparations as models of the human brain; (ii) using human peripheral tissue preparations as models of dynamic CNS processes; and (iii) studying human tissue, obtained postmortem, directly. Animal models are often readily obtained and reliable, and the high degree of inbreeding of common laboratory animals ensures that they usually yield consistent results. However, there are a number of human disorders for which animal models are either poor or unavailable, and species differences make extrapolation from the animal to the human case difficult. Human peripheral tissue models rely on a degree of homology between peripheral and CNS processes; in most cases, the evidence for such homologies derives from animal, rather than human, studies. Moreover, several examples are known where a peripheral process mimics the equivalent glial cell activity more closely than the neuronal, which can be a serious drawback for studies of neurotransmission. The use of postmortem human brain tissue presents a number of obvious difficulties, resulting from variations in the patient's age, agonal state, sex, preterminal medication, postmortem delay, etc. Human beings are genetically and nutritionally heterogeneous, so that data variability is usually greater here than when using tissue from laboratory animals. However, it is possible to control for a number of these factors, for example, by matching samples for basal metabolic rate and tissue integrity, and recently developed tissue freezing and storage techniques permit the use of within-subject experimental designs to help reduce experimental variation. A range of neurotransmitter functions are well retained in such tissue samples, so that regional variations, differential transmitter activities, drug effects, etc., can be studied in normal tissue samples, as well as in samples taken from cases of neurological and psychiatric disease. This allows, for example, changes in neuroanatomical indices to be correlated with localised alterations in a specific neurotransmitter function. A systematic approach to the analysis and matching of tissue samples is advocated. The three approaches should be considered to be complementary, especially for the study of human brain diseases.     

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.     

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.     

Isolation of alveolar epithelial cells from lung tissue obtained at autopsy

Summary Lung alveolar epithelial cells have been studied in a variety of laboratory animal models, and studies of human alveolar epithelial cells are important for comparison to information obtained from animal studies. Autopsy material is a source of human cells for study. Studies of human autopsy material revealed variables that negatively affected the yield of viable cells. For specimens from adults, these included death greater than 12 h before cell isolation, obvious severe lung fibrosis, longstanding metabolic disorders, and lung congestion indicated by weight of the right middle lobe greater than 150 g. Samples from children yielded significant numbers of viable cells up to 18 h after death. For 17 specimens that conformed to the above criteria, approximately 8.5×10⁶ alveolar cells were obtained per gram of tissue (tissue weights ranged from 30 to 108 g) using a procedure involving instillation of proteases into the airways. The cells could be further fractionated, and 10 to 15% of the mixed cells obtained were type II pneumocytes. Analysis of NADPH cytochrome-c-reductase distribution in subcellar fractions provided evidence that the cells obtained were intact. Phospholipid enzyme activities and synthetic activity were within the ranges previously found in laboratory studies of freshly obtained animal lungs. These results suggest that significant numbers of viable and functional human lung cells, including type II pneumocytes, can be obtained from autopsy material. This research was supported by a grant (HL 33083) from the National Heart, Lung and Blood Institute, Bethesda, MD.     

HUNTINGTON''S CHOREA: POST MORTEM ACTIVITY OF ENZYMES INVOLVED IN CEREBRAL GLUCOSE METABOLISM

Abstract— The loss of at least two different cell types in the basal ganglia of the choreic brain led us to examine the activity of enzymes involved in the metabolism of glucose. Cellular ATPase, HK, G6-PDH, PFK., LDH, GDH were measured. Post mortem stability studies indicated that these enzymes were more unstable in human brain than mouse brain. The most stable enzyme was GDH. HK activity appeared to increase after freezing, suggesting release from another compartment. PFK and G6-PDH activity decreased by 70% over the usual time and temperature period for autopsy. In the autopsied brain tissue we were still able to measure significant activities that allowed us to determine the distribution of these enzymes and the similar post mortem handling of control and choreic brain allowed us to compare these two groups.
The activity of HK and G6-PDH was higher in the frontal cortex than in the basal ganglia. Ouabain insensitive ATPase and PFK were higher in the basal ganglia than the frontal cortex.
GDH activity, an enzyme that is very active in glial cells, was increased in the choreic globus pallidus, an area with a very high glial to neuronal cell ratio.
Although there was a wide variation in PFK activity that appeared to be related to the pre-mortem clinical state there was a significant decrease in PFK activity in the putamen of choreic post mortem brain when compared to controls.
These findings do not indicate an absolute defect in any of the enzymes studied in choreic brain but further studies might prove worthwhile.     

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.     

Molecular forms of somatostatin, substance P and neurotensin in fresh human cerebral cortex

The molecular forms of somatostatin, substance P and neurotensin in fresh normal human cerebral cortex have been investigated using high performance liquid chromatography and radioimmunoassay. For each peptide most of the immunoreactivity measured corresponds to a single molecular form co-eluting with the authentic peptide. Small differences in the minor peaks of somatostatin and substance P immunoreactivity are seen when compared to the results of similar studies on post mortem human brain. A substantial difference in the molecular forms of neurotensin was seen which suggests that degradation of this peptide may occur post mortem.     

Partitioning the proteome: phase separation for targeted analysis of membrane proteins in human post-mortem brain

Neuroproteomics is a powerful platform for targeted and hypothesis driven research, providing comprehensive insights into cellular and sub-cellular disease states, Gene × Environmental effects, and cellular response to medication effects in human, animal, and cell culture models. Analysis of sub-proteomes is becoming increasingly important in clinical proteomics, enriching for otherwise undetectable proteins that are possible markers for disease. Membrane proteins are one such sub-proteome class that merit in-depth targeted analysis, particularly in psychiatric disorders. As membrane proteins are notoriously difficult to analyse using traditional proteomics methods, we evaluate a paradigm to enrich for and study membrane proteins from human post-mortem brain tissue. This is the first study to extensively characterise the integral trans-membrane spanning proteins present in human brain. Using Triton X-114 phase separation and LC-MS/MS analysis, we enriched for and identified 494 membrane proteins, with 194 trans-membrane helices present, ranging from 1 to 21 helices per protein. Isolated proteins included glutamate receptors, G proteins, voltage gated and calcium channels, synaptic proteins, and myelin proteins, all of which warrant quantitative proteomic investigation in psychiatric and neurological disorders. Overall, our sub-proteome analysis reduced sample complexity and enriched for integral membrane proteins by 2.3 fold, thus allowing for more manageable, reproducible, and targeted proteomics in case vs. control biomarker studies. This study provides a valuable reference for future neuroproteomic investigations of membrane proteins, and validates the use Triton X-114 detergent phase extraction on human post mortem brain.     

Biochemical and molecular studies using human autopsy brain tissue

The use of human brain tissue obtained at autopsy for neurochemical, pharmacological and physiological analyses is reviewed. RNA and protein samples have been found suitable for expression profiling by techniques that include RT-PCR, cDNA microarrays, western blotting, immunohistochemistry and proteomics. The rapid development of molecular biological techniques has increased the impetus for this work to be applied to studies of brain disease. It has been shown that most nucleic acids and proteins are reasonably stable post-mortem. However, their abundance and integrity can exhibit marked intra- and intercase variability, making comparisons between case-groups difficult. Variability can reveal important functional and biochemical information. The correct interpretation of neurochemical data must take into account such factors as age, gender, ethnicity, medicative history, immediate ante-mortem status, agonal state and post-mortem and post-autopsy intervals. Here we consider issues associated with the sampling of DNA, RNA and proteins using human autopsy brain tissue in relation to various ante- and post-mortem factors. We conclude that valid and practical measures of a variety of parameters may be made in human brain tissue, provided that specific factors are controlled.     

脑胶质瘤动物模型的研究及应用进展

脑胶质瘤约占中枢神经肿瘤的一半,临床治疗效果差。尤其是胶质母细胞瘤,其恶性程度极高,预后性差,是威胁人类健康的主要恶性肿瘤之一,因此,选择一种有效的动物模型是研究脑胶质瘤发病机制及其治疗方法的关键。随着分子生物学、遗传学的发展,尤其是转基因小鼠,以及其他越来越多模式生物的出现,目前已建立了多种脑胶质瘤动物模型。该文将对目前所建立的各种脑胶质瘤动物模型予以综述。     

The LPS receptor, CD14, in experimental autoimmune encephalomyelitis and multiple sclerosis.

Innate immune receptors are crucial for defense against microorganisms. Recently, a cross-talk between innate and adaptive immunity has been considered. Here, we provide first evidence for a role of the key innate immune receptor, LPS receptor (CD14) in pathophysiology of experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis. Indicating a functional importance in vivo, we show that CD14 deficiency increased clinical symptoms in active experimental autoimmune encephalomyelitis. Consistent with these observations, CD14 deficient mice exhibited a markedly enhanced infiltration of monocytes and neutrophils in brain and spinal cord. Moreover, we observed an increased immunoreactivity of CD14 in biopsy and post mortem brain tissues of multiple sclerosis patients compared to age-matched controls. Thus, the key innate immune receptor, CD14, may be of pathophysiological relevance in experimental autoimmune encephalomyelitis and multiple sclerosis.     

Coenzyme Q10 and its putative role in the ageing process

Summary A phenomenon associated with the aging process is a general age-dependent decline in cellular bioenergetic capacity that varies from tissue to tissue and even from cell to cell within the same tissue. This variation eventually forms a tissue bioenergy mosaic. Recent evidence by our group suggests that the accumulation of mitochondrial DNA mutations, in conjunction with a concurrent decrease in full-length mtDNA in tissues such as skeletal and cardiac muscle, strongly correlates with decreased mitochondrial function and accounts for the bioenergy mosaic. Evidence is also presented suggesting that amelioration with coenzyme Q₁₀ may restore some of the age-associated decline in bioenergy function, in effect providing the potential for a “redox therapy”. Coenzyme Q is a naturally occurring material that is present in the membranes of all animal cells. Its primary function is to act as an electron carrier in the mitochondrial electron transport chain enabling the energy from substrates such as fats and sugars (in the form of reducing equivalents) to be ultimately captured in the form of ATP, which in turn may be utilised as a source of cellular bioenergy. Coenzyme Q₁₀ has no known toxic effects and has been used in a limited number of animal studies and human clinical trials; however, the mechanism of action of coenzyme Q₁₀ remains unclear. A series of experiments by this group aimed at determining the efficacy of coenzyme Q₁₀ treatment on ameliorating the bioenergy capacity at the organ and cellular level will also be reviewed.     

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.     

Unconventional translation initiation of human trypsinogen 4 at a CUG codon with an N-terminal leucine. A possible means to regulate gene expression

Chromosomal rearrangements apparently account for the presence of a primate-specific gene (protease serine 3) in chromosome 9. This gene encodes, as the result of alternative splicing, both mesotrypsinogen and trypsinogen 4. Whereas mesotrypsinogen is known to be a pancreatic protease, neither the chemical nature nor biological function of trypsinogen 4 has been explored previously. The trypsinogen 4 sequence contains two predicted translation initiation sites: an AUG site that codes for a 72-residue leader peptide on Isoform A, and a CUG site that codes for a 28-residue leader peptide on Isoform B. We report studies that provide evidence for the N-terminal amino acid sequence of trypsinogen 4 and the possible mechanism of expression of this protein in human brain and transiently transfected cells. We raised mAbs against a 28-amino acid synthetic peptide representing the leader sequence of Isoform B and against recombinant trypsin 4. By using these antibodies, we isolated and chemically identified trypsinogen 4 from extracts of both post mortem human brain and transiently transfected HeLa cells. Our results show that Isoform B, with a leucine N terminus, is the predominant (if not exclusive) form of the enzyme in post mortem human brain, but that both isoforms are expressed in transiently transfected cells. On the basis of our studies on the expression of a series of trypsinogen 4 constructs in two different cell lines, we propose that unconventional translation initiation at a CUG with a leucine, rather than a methionine, N terminus may serve as a means to regulate protein expression.     

NPY and VGF immunoreactivity increased in the arcuate nucleus, but decreased in the nucleus of the Tractus Solitarius, of type-II diabetic patients

Ample animal studies demonstrate that neuropeptides NPY and α-MSH expressed in Arcuate Nucleus and Nucleus of the Tractus Solitarius, modulate glucose homeostasis and food intake. In contrast is the absence of data validating these observations for human disease. Here we compare the post mortem immunoreactivity of the metabolic neuropeptides NPY, αMSH and VGF in the infundibular nucleus, and brainstem of 11 type-2 diabetic and 11 non-diabetic individuals. α-MSH, NPY and tyrosine hydroxylase in human brain are localized in the same areas as in rodent brain. The similar distribution of NPY, α-MSH and VGF indicated that these neurons in the human brain may share similar functionality as in the rodent brain. The number of NPY and VGF immuno positive cells was increased in the infundibular nucleus of diabetic subjects in comparison to non-diabetic controls. In contrast, NPY and VGF were down regulated in the Nucleus of the Tractus Solitarius of diabetic patients. These results suggest an activation of NPY producing neurons in the arcuate nucleus, which, according to animal experimental studies, is related to a catabolic state and might be the basis for increased hepatic glucose production in type-2 diabetes.     

Concentration of Nucleosides and Related Compounds in Cerebral and Cerebellar Cortical Areas and White Matter of the Human Brain

1. Nucleosides potentially participate in the neuronal functions of the brain. However, their distribution and changes in their concentrations in the human brain is not known. For better understanding of nucleoside functions, changes of nucleoside concentrations by age and a complete map of nucleoside levels in the human brain are actual requirements.2. We used post mortem human brain samples in the experiments and applied a recently modified HPLC method for the measurement of nucleosides. To estimate concentrations and patterns of nucleosides in alive human brain we used a recently developed reverse extrapolation method and multivariate statistical analyses.3. We analyzed four nucleosides and three nucleobases in human cerebellar, cerebral cortices and in white matter in young and old adults. Average concentrations of the 308 samples investigated (mean±SEM) were the following (pmol/mg wet tissue weight): adenosine 10.3±0.6, inosine 69.5±1.7, guanosine 13.5±0.4, uridine 52.4±1.2, uracil 8.4±0.3, hypoxanthine 108.6±2.0 and xanthine 54.8±1.3. We also demonstrated that concentrations of inosine and adenosine in the cerebral cortex and guanosine in the cerebral white matter are age-dependent.4. Using multivariate statistical analyses and degradation coefficients, we present an uneven regional distribution of nucleosides in the human brain. The methods presented here allow to creation of a nucleoside map of the human brain by measuring the concentration of nucleosides in microdissected tissue samples. Our data support a functional role for nucleosides in the brain.     

人肺组织特性区域差异的实验方法初探

对非肺病因死亡的病人在24小时内解剖而得的人肺各叶进行了研究,以了解人肺组织弹性的区域性差异。人肺矩形薄片试件在二维试验机上进行试验。结果表明:在一定肺容量下,右上叶弹性模量为最大,依次为左上叶,右下叶,左下叶和右中叶。从增量模量观点看、人肺呈现相当大的区域差异。     

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.