Christoph Meissner

The 4977 bp deletion of mitochondrial DNA in human skeletal muscle, heart and different areas of the brain: a useful biomarker or more?

It has been suggested that deletions of mitochondrial DNA (mtDNA) are important players with regard to the ageing process. Since the early 1990s, the 4977 bp deletion has been studied in various tissues, especially in postmitotic tissues with high energy demand. Unfortunately, some of these studies included less than 10 subjects, so the aim of our study was to quantify reliably the deletion amount in nine different regions of human brain, heart and skeletal muscle in a cohort of 92 individuals. The basal ganglia contain the highest deletion amounts with values up to 2.93% and differences in deletion levels between early adolescence and older ages were up to three orders of magnitude. Values in frontal lobe were on average an order of magnitude lower, but lowest in cerebellar tissue where the amount was on average only 5 x 10(-3) of the basal ganglia. The deletion started to accumulate in iliopsoas muscle early in the fourth decade of life with values between 0.00019% and 0.0035% and was highest in a 102-year-old woman with 0.14%. In comparison to skeletal muscle, the overall abundance in heart muscle of the left ventricle was only one-third. The best linear logarithmic correlation between amount of the deletion and age was found in substantia nigra with r=0.87 (p<0.0005) followed by anterior wall of the left ventricle (r=0.82; p<0.0005). With regard to mitochondrial DNA damage, we propose to use the 4977 bp deletion as an ideal biomarker to discriminate between physiological ageing and accelerated ageing. The biological meaning of mitochondrial deletions in the process of ageing is under discussion, but there is experimental evidence that large-scale deletions impair the oxidative phosphorylation in single cells and sensitize these cells to undergo apoptosis.

Lack of age-related increase of mitochondrial DNA amount in brain, skeletal muscle and human heart

During the ageing process, an increase of mitochondrial DNA (mtDNA) deletions and other mutations have been reported. These structural alterations of mtDNA are assumed to cause a reduction in the respiratory chain activity and may contribute to the ageing process. Therefore, the question arises if the accumulation of deleted mtDNA is compensated in vivo by an increase of mtDNA synthesis via a feedback mechanism. We designed two human mtDNA-specific oligonucleotide probes for quantitative mtDNA analysis of 5 different tissues from 50 individuals aged from 8 weeks to 93 years. The amount of mtDNA was approximately 1.1 +/- 0.5% (4617 +/- 2099 copies) in the caudate nucleus, 1.0 +/- 0.5% (4198 +/- 2099 copies) in the frontal lobe cortex, 0.3 +/- 0.2% (1259 +/- 840 copies) in the cerebellar cortex, 1.0 +/- 0.4% (4198 +/- 1679 copies) in skeletal muscle and 2.2+/-1.3% (9235 +/- 5457 copies) in heart muscle. We did not observe any significant change in the absolute copy number during ageing in five different tissues, and therefore, found no evidence for the postulated feedback mechanism. Our study indicates that mtDNA copy number is tissue-specific and depends on the energy demand of the tissue.

Tissue-specific deletion patterns of the mitochondrial genome with advancing age.

Aging is a multifactorial process and a lot of theories have been put forward to explain the deterioration of organ function with advancing age. The free radical hypothesis developed by Harman is amongst the most prominent today and has been focused on mitochondrial aging in the last decades. Applying a long PCR approach we screened human skeletal muscle, heart, caudate nucleus and cerebellum of 50 individuals for large-scale deletions of mitochondrial DNA (mtDNA). The most important observation of our study was the detection of age dependent tissue specific deletion patterns of mtDNA. The pattern of the same tissue of different individuals was more similar than the pattern of different tissues of the same individuals. Whereas deletions were barely detectable in cerebellar tissue, in caudate nucleus a specific banding pattern with deletions of 4-8 kb was already observed around the age of thirty. However, the increase of these large-scale deletions in number and variety over lifetime was more pronounced in skeletal muscle or heart. Our data support the notion that different tissues accumulate mtDNA damage in a specific manner. Although functional consequences of mitochondrial deletions are clearly supported by experimental data on the single-cell level in model organisms and mammals, their role regarding impaired function of organs with advancing age in humans remains unresolved.

Time Course of Cortical Hemorrhages after Closed Traumatic Brain Injury: Statistical Analysis of Posttraumatic Histomorphological Alterations

We examined 305 autopsied brains for histomorphological alterations to determine the time course of reactions in cortical hemorrhages following traumatic closed brain injury. Eighteen morphological criteria were considered: red blood cells (RBCs), polymorphonuclear leukocytes (PMNs), macrophages (Ms), RBC-containing Ms, hemosiderin, hematoidin, lipid-containing Ms, fibroblasts, endothelial cells, collagenous fibres, gemistocytic astrocytes, fibrillary gliosis, hemosiderin-containing astrocytes, neuronal damage, neuronophagy, axonal swelling (beta-amyloid precursor protein: beta-APP), axonal bulbs (van Gieson stain), and mineralisation of neurons. The interval between the time of brain injury and death ranged from 1 min to 58 years. Following routine staining and immunohistochemical staining of microglia (CD68), astrocytes (GFAP) and injured axons (beta-APP), paraffin sections were examined by light microscopy for the presence of the selected histomorphological features. For each cytomorphological phenomenon, the time at which it could be demonstrated for the first time and for the last time (observation period) was determined. The relative frequency of each criterion was established for each observation period. The limits of confidence for the respective relative frequencies were estimated with a reliability of 95% according to Clopper and Pearson. An apparent correlation was found between the frequency of a given histomorphological phenomenon and the length of the posttraumatic interval. To check for accuracy of prediction, half of the cases (group 1; n = 153) were used to develop a multistage evaluation model; half (group 2; n = 152) were used to evaluate the validity of the data of group 1. Applying this model, 117 of the 152 control group cases (76.97%) could be correctly classified and further 26 cases (17.11%) being assigned to an interval close to the correct interval. Thus, this model allows classification of the correct posttraumatic interval or an interval close to the correct posttraumatic interval in about 95% of cases. We developed a software program that allows the estimation of survival time of TBI based on the relative frequency of the 18 morphological features. Applying this software will help to estimate the posttraumatic interval of cortical hemorrhages following TBI of unknown survival time.

Estimation of age at death based on quantitation of the 4977-bp deletion of human mitochondrial DNA in skeletal muscle.

The 4977-bp deletion in human mitochondrial DNA (mtDNA) is known to accumulate in various tissues with age. Since this deletion in mtDNA correlates closest with age in muscle tissue, iliopsoas muscle tissue was taken at autopsy from 50 persons aged 24-97 years to determine whether age at death can be estimated based on the amount of the 4977-bp deletion in skeletal muscle. Total DNA (nuclear and mtDNA) was extracted from 100 mg tissue and the 4977-bp deletion quantified using a kinetic polymerase chain reaction (PCR) followed by visualization of the products on silver stained polyacrylamide gels. The amount of the 4977-bp deletion of mtDNA ranged from 0.00049% to 0.14% depending on age, with a correlation coefficient of r = 0.83 (P = 0.0001). In forensic practice this method can aid in the estimation of age at death with a relatively wide confidence interval, thus enabling a discrimination between young and elderly persons in the identification of human remains based solely on skeletal muscle.

Quantification of mitochondrial DNA in human blood cells using an automated detection system.

The 4977 bp deletion of mitochondrial DNA (mtDNA) accumulates in postmitotic tissues with advancing age. The purpose of our study was to detect and quantify these deletion even in blood cells with a high turnover activity. Whole venous blood, isolated human platelets and peripheral blood mononuclear cells (PBMCs) were collected from 10 unrelated donors aged 20-71 years and total DNA was extracted. PCR was performed for total and mutated mtDNA using two different primer pairs and two fluorogenic probes labeled with the fluorescent dyes FAM and VIC. Specific PCR products were generated, detected and quantified in a real-time PCR. The amplification products of total and deleted mtDNA could be detected in each sample and did not exhibit any differences in the amount of the deleted mtDNA in whole blood, human platelets or PBMCs. Our data did not show any accumulation of the 4977 bp deletion with increasing age as it was observed for several other tissues.

Demonstration of the 4977 bp deletion in human mitochondrial DNA from intravital and postmortem blood

The 4977 bp deletion in mitochondrial DNA (mtDNA) is known to accumulate with age in various human tissues. Findings regarding its accumulation in blood, however, have so far been contradictory. We investigated the levels of the 4977 bp deletion in mtDNA from 100 intravital and postmortem blood samples. Applying an improved version of a PCR plus silver staining of polyacrylamide gels, we could detect the 4977 bp deletion in blood of healthy individuals over 20 years of age. While the 4977 bp deletion in blood is subject to a certain age dependence, it appears to be influenced by additional factors. A Primer-Shift-Assay amplifying four different deletion-specific fragments showed that the smaller fragments were amplified with a higher amplification efficiency than the larger fragments. The deletion-specific 389 bp fragment was demonstrated in 73% of individuals over 80 years of age, but in only 46% of individuals between 21 and 30 years old whereas the largest 802 bp deletion-specific fragment was detectable in 38% of subjects over 80 years of age, and in only 15% of individuals under 30 years of age. Deletion-specific fragments were not detected in a single individual under 20 years old, nor in fetal blood. In this work, we demonstrate for the first time the detection of 4977 bp specific fragments in blood of healthy individuals without the necessity of using a nested PCR. The deletion is detectable in postmortal and intravital blood, so that the occurrence of the 4977 bp deletion seems to be a physiological and not only a postmortal process.

Fatal versus non-fatal heroin "overdose": blood morphine concentrations with fatal outcome in comparison to those of intoxicated drivers

The study was performed to distinguish fatal from non-fatal blood concentrations of morphine. For this purpose, blood levels of free morphine and total morphine (free morphine plus morphine conjugates) in 207 cases of heroin-related deaths were compared to those in 27 drivers surviving opiate intoxication. The majority of both survivors and non-survivors were found to show a concomitant use of depressants including alcohol or stimulants. Blood morphine levels in both groups varied widely, with a large area of overlap between survivors (free morphine: 0-128 ng/ml, total morphine: 10-2,110 ng/ml) and non-survivors (free morphine: 0-2,800 ng/ml, total morphine: 33-5,000 ng/ml). Five (18.5%) survivors and 87 (42.0%) non-survivors exhibit intoxication only by morphine. In these cases, too, both groups overlapped (survivors-free morphine: 28-93 ng/ml, total morphine: 230-1,451 ng/ml; non-survivors-free morphine: 0-2,800 ng/ml, total morphine: 119-4,660 ng/ml). Although the blood levels of free or total morphine do not allow a reliable prediction of survival versus non-survival, the ratio of free/total morphine may be a criterion to distinguish lethal versus survived intoxication. The mean of the ratio of free to total morphine for all lethal cases (N=207) was 0.293, for those that survived (N=27) 0.135, in cases of intoxication only by morphine 0.250 (N=87) and 0.080 (N=5), respectively. Applying a cut-off of 0.12 for free/total morphine and performing ROC analyses, fatal outcome can be predicted in 80% of the cases correctly, whereas 16% of the survivors were classified as dead. Nevertheless, in this study, all cases with a blood concentration of 200 ng/ml and more of free morphine displayed a fatal outcome.

Detection of the 4977 bp deletion of mitochondrial DNA in different human blood cells

As recently reported, it is possible to detect and quantify the amount of the deleted human mitochondrial DNA (mtDNA) in whole blood, platelets and peripheral blood mononuclear cells using real-time PCR. The aim of this study was to identify the cell types in human blood carrying the 4977 bp deleted mtDNA and their accumulation with regard to donor age. Whole blood from 10 healthy donors (five individuals aged from 19 to 22 years, five aged from 57 to 61 years) was separated in various cell populations such as granulocytes, B cells/monocytes and T cells. Purity of the cell isolates was determined by flow cytometry. Total DNA was extracted and 250 ng DNA of each cell type was subjected to PCR using fluorescent-labelled primer pairs. The specific PCR product of the 4977 bp deletion was quantified using an automated detection system. The accumulation of the 4977 bp deletion was more pronounced in T lymphocytes and granulocytes in comparison to B lymphocytes/monocytes. The amount of the 4977 bp deletion in whole blood varied from 0 to 0.00018%, in T lymphocytes from 0.00009 to 0.00160%, in granulocytes from 0 to 0.00162% and in the B lymphocyte/monocyte fraction from 0 to 0.00025%. The higher amount of the deletion in T lymphocytes may be due to a subset of lymphocytes with a longer lifespan thus facilitating the accumulation of mitochondrial damage. The higher amount in granulocytes could have the explanation in the higher release of free radicals for prevention of infectious diseases, because free radicals are supposed to damage the macromolecules of this cell type. The 10 donors displayed differences in the pattern of the accumulation with regard to the different cell types, but no age-dependent accumulation was observed. Differences of the accumulation pattern may be due to actual individual living behaviour or environmental factors.

Microtubule-associated protein 2 (MAP2)—a promising approach to diagnosis of forensic types of hypoxia-ischemia

The loss of neuronal immunoreactivity of the cytoskeletal microtubule-associated protein 2 (MAP2) is known to be a marker of—at least—transient functional failure of neurons following ischemia. Because there are no specific neuropathological findings in forensic types of acute hypoxia-ischemia, detection of this relevant cause of death is often complicated and a reliable ischemic biomarker would be of great importance. We therefore investigated the neuronal immunoreactivity of MAP2 in human cases of forensic significance. A control group (n=27) was compared to a group of cases of hypoxia-ischemia (n=45), comprising death due to hanging (n=19), drowning (n=14) and carbon monoxide (CO) poisoning (n=12). Using immunohistochemical staining, the percentage of MAP2-positive neurons in the hippocampus (areas CA1–CA4) and frontal cortex (layers II–VI) was evaluated and compared. The hypoxia-ischemia group showed decreased MAP2 immunostaining in the hippocampal areas CA2–CA4 (P<0.05) and in cortical layers II–VI (P<0.001) compared to controls. Most vulnerable regions seem to be the hippocampal CA4 area and cortical layers III–V. Within the hypoxia-ischemia group, death due to CO poisoning was characterized by the lowest MAP2 immunoreactivity. The hypoxic-ischemic groups differ from controls by a distinct decrease of MAP2 immunostaining. Thus, the loss of MAP2 immunoreactivity may support the diagnosis of neuronal injury in forensic types of hypoxia-ischemia, although investigations on postmortem tissue must be interpreted cautiously.