Mast cell reactivity at the margin of human skin wounds: an early cell marker of wound survival?

Detecting the vitality of mechanical skin wounds (antemortem versus postmortem injury) in human cadavers remains a specifically forensic problem. To determine whether skin mast cells (MCs) are activated during the very early phase of human wound healing we performed a histomorphometric evaluation of the extent of MC enzyme loss as an indication of MC degranulation at the wound margins of skin wounds in 64 human cadavers. We compared the number of tryptase-reactive MCs, which are said not to loose all of their enzyme activity during degranulation process, with the number of naphthol AS-D chloroacetate esterase (NAS-DClAE)-positive MCs, which loose their complete enzyme activity in the form of enzyme-positive granula after activation. The enzyme activity was evaluated on sequential histological sections after autopsy as an indirect quantification of the number of degranulated MCs. Most of the victims had died within 10-60 min after injury (n=50), 12 survived between 60 min and 24h, and only 2 victims survived more than 24h (12 days each). The number of enzyme-positive MCs were counted in six successive visual fields (0.785 mm(2)) on the one hand located parallel to and--on the other hand--at increasing distances outward from the wound margins. In victims surviving the injury less than 60 min the average number of NAS-DClAE-reactive MCs next to the wound margin was significantly lower than the number of tryptase-reactive MCs. The extent of the reduction in NAS-DClAE-reactive MC counts correlated inversely with the distance from the wound edges. Our findings show that MCs undergo very early loss of NAS-DClAE activity at wound margins, and thus appear to be an early cell marker of wound survival. However, definitive evidence that the enzyme loss (degranulation) represents a vital process can only be obtained by comparing MC enzyme loss induced by injury during intact circulation with the MC reaction to injury inflicted very shortly after cardiac arrest, a question that can only be answered by animal experiments.

Macrophage subsets in mechanical brain injury (MBI)--a contribution to timing of MBI based on immunohistochemical methods: a pilot study.

Cortical hemorrhages as a consequence of closed mechanical brain injury (MBI) trigger an inflammatory response including a distinct increase of macrophages. According to published data this reactive macrophage population is heterogenous as to their immunological properties. The expression of certain immunohistochemically detectable epitopes of macrophages, however, may correlate with the posttraumatic interval (PTI). In a pilot study, 50 selected cases of cortical hemorrhages with 1 min to 1.5 years PTI were examined by light microscopy and macrophages were labeled with CD68-, HLA-D-, HAM-56-, LN-5-, and 25F9-antibodies, while hemosiderin was detected by a Prussian-blue reaction. Qualitative and semiquantitative investigations were performed. The semiquantitative study included 5 different classes. The results of the study revealed a distinct timetable of the appearance of macrophages labeled with certain antibodies. While HLA-D immunoreactivity was detected after a PTI of 6h in the cortex and white matter bordering the traumatic hemorrhage, CD68 immunopositive macrophages were present after 12h, LN-5 and HAM-56 after 48h, and 25F9 within 10d. Hemosiderin-containing macrophages were detectable within 100h in the same region. Within the hemorrhage itself a certain immunoreactivity of macrophages starts several hours before: CD68 after 3h, LN-5 after 24h, HAM-56 after 31h, hemosiderin after 76h, and 25F9 after 4d. For forensic purposes these observations are of crucial importance because the time course of the appearance of certain immunopositive macrophages labeled with different antibodies allows a differentiated timing of contusional injuries; however, the cause of this different immunopositive reaction remains unexplained. The observed time dependency of different macrophage antigen expressions in cortical hemorrhages after closed head injury is a suitable method to estimate the PTI and will allow a forensic reliable estimation if future investigations are extended on higher numbers of cases and/or additional markers.

The effect of chronic alcohol consumption on mitochondrial DNA mutagenesis in human blood.

The 4977bp deletion of mitochondrial DNA (mtDNA) is known to accumulate with increasing age in post mitotic tissues. Recently, studies came out detecting this specific alteration also in fast replicating cells, e.g. in blood or skin tissue, often in correlation to specific diseases or -- specifically in skin -- external stressors such as UV radiation. In this study, we investigated mitochondrial mutagenesis in 69 patients with a chronic alcoholic disease and 46 age matched controls with a moderate drinking behavior. Two different fragments, specific for total and for deleted mtDNA (dmtDNA) were amplified in a duplex-PCR. A subsequent fragment analysis was performed and for relative quantification, the quotient of the peak areas of amplification products specific for deleted and total mtDNA was determined. Additionally, a real time PCR was performed to quantify mtDNA copy number. The relative amount of 4977bp deleted mtDNA in alcoholics was significantly increased compared to controls. On the other hand, no difference regarding the mtDNA/nuclear DNA ratio in both investigated groups was detected. Additionally, no age dependence could be found nor in alcoholics, neither in the control group. These findings indicate that mtDNA mutagenesis in blood can be influenced by stressors such as alcohol. Ethanol seems to be a significant factor to alter mitochondrial DNA in blood and might be an additional contributor for the cellular aging process.

A new sensitive short pentaplex (ShoP) PCR for typing of degraded DNA.

Analysis of short tandem repeat makers has become the most powerful tool for DNA typing in forensic casework analysis. Unfortunately, typing of DNA extracted from telogen shed hairs, bones buried in the soil or from paraffin-embedded, formalin-fixed tissue often reveals no results due to the degradation of DNA. The reduction in size of the target fragments by development of new primers and their combination in multiplex approaches open a new field of DNA analysis. Here we present a new sensitive short pentaplex PCR including the loci amelogenin, TH01, VWA, D3S1358 and D8S1179. Validation tests of our new method included sensitivity, mixtures, human specificity, artificial degradation of DNA by DNase I and case work analysis on a panel of different forensic samples. The detection limit was 12.5 pg of human DNA, and mixtures of 50 pg in a total of 1000 pg were clearly detectable and revealed complete profiles. Only DNA extracts of human primates displayed a few signals, whereas other animal, fungal or bacterial DNA showed no signals. Our method proved extremely valuable in the analysis of artificially degraded DNA and in forensic cases, where only poorly preserved DNA was available. This approach and other similar methods can aid in the analysis of samples where allelic drop out of larger fragments is observed. It is highly recommended to develop more of these multiplexes to improve poor quality DNA typing.

Fall or shaken: traumatic brain injury in children caused by falls or abuse at home - a review on biomechanics and diagnosis.

In cases of traumatic brain injuries in children it may be difficult to differentiate between physical abuse and accidental occurrence. This review will shed light on discriminating epidemiological as well as biomechanical data, morphological findings, and clinical features. As a basic principle, simple injuries are caused by simple mechanisms like falls whereas life-threatening injuries should be attributed to abuse until proven otherwise. Implausibilities between reported events and mechanisms by caregivers as well as more than one explanation of injury indicate suspicion of child abuse. From reviewing the literature, it can be stated that falls from less than 1.5 m lead only in few cases to severe brain injuries. Children who experienced a fall at home seldom exhibit fractures of the skull but just minor intracranial injuries without neurological deficits. Regarding biomechanical aspects, multiple or complex skull fractures, depression fractures, additional fractures of the body, and intracranial hemorrhages as a consequence of an impact are the most important findings in child abuse. Moreover, additional specific morphological criteria give evidence of clinical and/or post-mortem diagnosis of the shaken baby syndrome. These include subdural hemorrhage and laceration of the brain and retinal bleeding, epidural hemorrhage of the cervical cord, gripping marks (bruises) on chest and/or shoulders, and tearing injuries of the throat and neck muscles. Post-mortem signs of recurrent shaken baby syndrome are indicated by iron-positive cells or microglial cells in neuronal or retinal tissue. A clinical diagnosis will be dependent on the one hand on the exclusion of coagulation diseases, on the other hand on the demonstration of a subdural hemorrhage by means of neuroimaging techniques, i.e., CT and MRI, as well as retinal hemorrhage. The shaken baby syndrome will usually be observed within the first year of life. The doctor has to manifest his diagnosis to the caregivers and - before informing the caregivers - he must be sure of his diagnosis.

Gunshot injuries to the head and brain caused by low-velocity handguns and rifles. A review.

OBJECTIVES: Reconstruction of brain injuries is a basic task of forensic neuropathology. For better understanding of the wound ballistics of gunshot injuries to the brain caused by low-velocity firearms (E(o) < 550 J), we reviewed the respective contributions of: (1) biomechanical reconstruction by postmortem imaging techniques, (2) biometry of the extent of very early microscopic tissue destruction, and (3) microscopic studies on the type and extent of early microscopic reactions around the permanent missile track. MATERIAL AND METHODS: A selected case material of 47 victims of lethal gunshot wounding to the brain was studied. (1) Computed tomography (CT) and magnetic resonance imaging (MRI) techniques were compared with macroscopic findings in 17 cases. (2) Morphometric evaluation of the zones of cellular and axonal destruction around the permanent track was performed in 20 cases (survival time: <90 min). (3) Microscopic studies of the emigration of leukocytes and macrophages plus axonal expression of beta-amyloid precursor protein (beta-APP) were conducted in 10 cases (survival time: >90 min). RESULTS AND CONCLUSIONS: (1) Imaging procedures provided valuable information on entrance and exit wounds, the missile track and secondary changes. (2) Biometry revealed a destruction zone of ca. 3.6 cm around the permanent track corresponding to the "temporary cavity". (3) Microscopic studies of reactive changes demonstrated axonal injury at sites remote from the permanent cavity that could explain the very early respiratory arrest following low-velocity gunshot injury.

Vitality and time course of wounds.

The term "wound" describes the morphologic-functional disruption of the continuity of a tissue structure. A wound can be inflicted during life--when the cardiovascular and respiratory system is still intact--or after death, i.e. after cardiac and respiratory arrest. Traumatization during life triggers vital reactions that do not occur in postmortem wounds. Three types of vital reactions in wound healing can be distinguished: Reactions of the scavenger type, which are almost exclusively mediated by blood cells. Reactions by complex signal transduction pathways, which involves cascade-like release of chemokines, cytokines and adhesion molecules and may influence type 1 and type 3 reactions. Reactions of the scarring type, which involve the final repair of the damaged tissue and are carried out primarily by cells residing at the wound edges, i.e. partly concerning mesenchymal cells and partly tissue-specific cells dependent on the involved organ system. The three different types of reaction follow roughly parallel temporal courses that include cascade-like interactions among themselves. Whereas demonstration of a vital reaction suffices to differentiate an intravital wound from a postmortem wound, the vital reactions themselves follow strictly temporal courses. The regular time-dependent occurrence of each phenomenon allows--in limits--a reliable temporal classification of wound healing. A review will be given especially demonstrating the actual German scientific research in vitality and in skin wound timing as well as in timing of mechanical injury of the brain.

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.

Forensic pathological aspects of postmortem imaging of gunshot injury to the head: documentation and biometric data.

To determine the value of imaging procedures such as computer tomography (CT) and magnetic resonance imaging (MRI) of the head in providing additional information of forensic relevance, we examined 17 cadavers of human victims of gunshot wounds to the head. Three of the victims briefly survived the gunshot wound. The weapons involved were all guns with low muzzle energy (<550 J), i.e., handguns and low-velocity rifles. In the majority of cases ( n=15) a penetrating wound to the head was found, only two cases showed the bullet lodged in the brain. In some cases, imaging of the skull and brain was performed prior to autopsy; in others imaging took place after autopsy on the isolated, formalin-fixed brain. The imaging findings were correlated with the criminological data and the results of macroscopic and microscopic examination of the brain. The findings on the bony structures of the head provided imaging criteria for differentiation between entrance and exit of the gunshot wounds, which corresponded to the forensic pathological findings at autopsy. CT scans and MRI of the cerebral parenchyma revealed lanes of opaque bone and missile fragments along the course of the missile, which allowed recognition of the missile track in 3D reconstruction. Biometric reconstruction allowed easy determination of the angle of the missile track in all three planes. Examination of the parenchymal structures and imaging of the isolated, formalin-fixed brain enabled tracking of the missile path directly along the zone of destruction as well as demonstration of secondary changes such as air bubbles along the bullet course, hemorrhage and edema. The significance of a translucent zone surrounding the missile track in several cases remains unclear; it probably represents tissue destruction secondary to temporary cavitation. The imaging procedures described here allowed excellent documentation of in situ conditions, while the storing of data enabled biometric reconstruction for determination of the angle of trajectory, of entrance and exit wounds, and the extent of tissue damage along the missile track and, possibly, in the zone of temporary cavitation.

Quantification of human mitochondrial DNA in a real time PCR.

Recently, a moderately priced machine for real-time quantitative PCR has become available, the Perkin Elmer 5700. The rapid and quantitative assay of mitochondrial DNA (mtDNA) copy number is potentially useful in a variety of molecular, evolutionary and forensic fields. Using this new tool, we have evaluated the precision and reliability of the real time PCR to quantify undeleted mitochondrial genome copy number, and to determine the frequency of an age-associated deletion of 4977 base pairs in length, in 42 human iliopsoas muscle DNA samples from persons of known age. We have evaluated the accuracy with which age can be predicted, knowing only the frequency of this common 4977 bp deletion, and derived a statistical formula which describes the confidence with which the 4977 bp frequency predicts age. The results indicate that the mutation frequency could be used to distinguish between tissue from young and old individuals. However in this data set, while there was considerable agreement of 4977 bp frequency among replicates from the same individual sample, there was substantial diversity of mean mutation frequency between individuals of the same or similar ages. The simplest interpretation of these results is that there are biological modifiers of 4977 bp frequency that are age-independent, which are potentially interesting but may limit the usefulness of this deletion frequency alone as a "molecular forensic clock."

Mitotic brain cells are just as prone to mitochondrial deletions as neurons: a large-scale single-cell PCR study of the human caudate nucleus.

Mitochondria are considered a key element in the process of organismic aging, because of their fundamental role in cellular energy generation. In the course of oxidative phosphorylation, harmful free radicals are continuously produced damaging the mitochondrial (mt) genome. One of the consequences is the occurrence of large-scale deletions in mtDNA molecules. The 4977 bp common deletion accumulates exponentially with age, in a mosaic pattern, especially in postmitotic tissues. In order to investigate whether certain cell characteristics underlie this pattern of distribution, and to look for possible age-related changes, two cell types in the caudate nucleus of the human brain from five young and five senescent subjects were analysed by single-cell PCR.MAP2-positive neurons and GFAP-positive astrocytes were isolated by micromanipulation. For each of the 10 cases, at least 30 cells of each type were collected and subjected to PCR individually. Screening for the presence of the common deletion yielded no significant differences in relative distribution, neither between astrocytes and neurons, nor between healthy young and old humans. Our results imply that the age-dependent increase of the common deletion cannot come about by an increase of independent deletion events in a greater proportion of cells, and that mitotic rate is not a major cellular risk factor for deletion accumulation in the caudate nucleus.

Proliferative activity of epidermal basal cells after wounding. AgNOR counts compared with bromodeoxyuridine reactivity in rats.

Quantitative changes in nucleolus organizer regions (NORs) are known markers of proliferation that can be demonstrated by a specific silver staining technique on paraffin-embedded sections. Wounding of skin induces proliferation of basal epidermal cells at the wound margin. The degree of proliferation depends on the survival time and can be measured by morphometric assessment of argyrophilic NORs (AgNORs). Following incision wounding of the pinnae, rats were allowed to survive for different intervals (7 rats per interval) up to 120 hours. Before each sacrifice, biopsies were taken and incubated in a bromodeoxyuridine (BrdU) solution, embedded in paraffin, and stained with an antibody against BrdU. At the same time morphometric analysis of AgNOR counts was performed on sections made from the same material. BrdU incorporating nuclei were assessed by simple counting, whereas morphometric analysis of AgNOR counts was computer aided. Both methods revealed an increase in the number of proliferating cells, a plateau phase being reached after about 36 hours, followed by a decline after about 70 hours. Both methods thus allowed a reliable temporal classification of the skin injury according to survival time. The molecular background of the AgNOR changes in relation to the proliferation of cellular elements is discussed in detail.

Creutzfeldt-Jakob disease (CJD) in a case of suspected chronic heavy metal poisoning.

We describe a patient who died of suspected heavy metal poisoning after a nine-month history of rapidly worsening dementia. Autopsy at a forensic-pathological institute established the postmortem diagnosis of sporadic Creutzfeldt-Jakob disease (CJD) based on demonstration of the proteinase-resistant prion protein (PrPsSc) in Western-Blot on native brain tissue. Microscopic examination of the macroscopically largely inconspicuous brain revealed marked spongiform changes in the gray matter--mainly affecting the cerebral cortex, nucleus caudatus, and putamen--with confluent vacuoles. Patchy or perivacuolar deposits of PrPSc were found as well as granular PrPsc deposits. The cerebellum contained focal PrPsc deposits. There was an astrogliosis in the white matter and a proliferation of microglia in the gray matter with a simultaneous clear reduction in neuronal elements. The differential diagnosis is discussed, as is the potential risk to those performing autopsy on forensic cases with a clinical picture of rapidly progressing dementia, especially in cases where a prion disease is not initially suspected.

Time of drug elimination in chronic drug abusers. Case study of 52 patients in a "low-step" detoxification ward.

The elimination time of illicit drugs and their metabolites is of both clinical and forensic interest. In order to determine the elimination time for various drugs and their metabolites we recruited 52 volunteers in a protected, low-step detoxification program. Blood samples were taken from each volunteer for the first 7 days, daily, urine sample for the first 3 weeks, daily. Urine was analyzed using a fluorescence-polarization immunoassay (FPIA) and gas chromatography/mass spectrometry (GC/MS), serum using GC/MS. The elimination times of the drugs and/or their metabolites in urine and serum as well as the tolerance intervals/confidence intervals were determined. Due to the sometimes extremely high initial concentrations and low cut-off values, a few of the volunteers had markedly longer elimination times than those described in the literature. The cut-off values were as follows: barbiturates II (200ng/ml), cannabinoids (20ng/ml), cocaine metabolites (300ng/ml), opiates (200ng/ml). GC/MS detected the following maximum elimination times: total morphine in urine up to 270.3h, total morphine and free morphine in serum up to 121.3h, monoacetylmorphine in urine up to 34.5h, 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-COOH) in urine up to 433.5h, THC-COOH in serum up to 74.3h, total codeine in urine up to 123h, free codeine in urine up to 97.5h, total codeine in serum up to 29h, free codeine in serum up to 6.3h, total dihydrocodeine (DHC) in urine up to 314.8h, free DHC in urine up to 273.3h, total and free DHC in serum up to 50.1h. Cocaine and its metabolites were largely undetectable in the present study.

Regional potassium distribution in the brain in forensic relevant types of intoxication preliminary morphometric evaluation using a histochemical method.

A histochemical-morphometric method was used to measure potassium (K+) levels in gray and white matter of rats following sublethal intoxication with 11 different neurotoxic compounds of high forensic significance. Six rats were each given a single substance applied intraperitoneally, the same dosage being given to two animals each. The animals were subsequently killed, the brains immediately frozen, and cryosections cut. K+ levels were evaluated morphometrically. A drop in K+ levels was used as the criterion for cytotoxic edema. Application of ethanol, atropine, carbromal, carbon monoxide, morphine or triethyltin led to a rise in K+ levels in the gray matter and a simultaneous decline in the white matter. By contrast, administration of amitriptyline, glycerin, potassium cyanide, parathion or phenobarbital initiated an increase in K+ levels in both gray and white matter. A cytotoxic edema could thus be reliably excluded in these intoxications. Although the study design allows no statistical analysis, these conclusions are supported by the marked differences in K+ levels in gray and white matter induced by the different toxicants.

Death from thermal effects and burns.

One hundred and fifteen unselected autopsy cases of death from thermal effects and/or fire between 1990 and 1999 were analyzed with regard to time of death, signs of vitality at the scene of the fire, the manner and cause of death, and the extent of soft tissue loss. The cases represented approximately 6% of all autopsy cases at the Institute of Legal Medicine responsible for a catchment area with approximately 700,000 inhabitants. In 23 victims suffering burn injuries, death occurred during initial medical care or clinical treatment. The causes of death were primary respiratory arrest due to smoke poisoning or delayed shock caused by thermal injuries to the skin. Death occurred at the scene of the fatal event in 85 cases: eight cases exhibited no thermal effects; the cause of death in one of these cases was polytrauma incurred in a leap from a height; in seven cases there was poisoning due to smoke inhalation. The remaining 77 cases were characterized by signs of intensive thermal and/or fire effects. Clear signs of vitality (carboxyhemoglobin (COHb) in blood, inhalation and/or swallowing of soot) were found in 84.7% of the victims dying at the site of the fatal event. Of the 13 victims showing no signs of vitality at the scene, a cause of death could be determined in only seven cases; death in the other six cases remains unexplained. Quantification of the soft tissue loss revealed a possible correlation with the temperature and time course of heat exposure.

Brain injury after survived gunshot to the head: reactive alterations at sites remote from the missile track.

Gunshot wounds to the brain usually lead to acute respiratory arrest or death after a brief survival period, even in cases involving only slight direct tissue damage. It can be assumed therefore that the damage extends beyond the zone of recognizable destruction and hemorrhages. To determine the true extent of the tissue injury resulting from gunshot wounds to the brain, we carried out microscopic investigations for reactive changes (emigration of leukocytes and macrophages, axonal expression of beta-amyloid precursor protein (beta-APP) in 10 cases of gunshot wound to the narrow channel of the brain with survival times >2h. Demonstration of leukocytes expressing naphthol AS-D chloroacetate esterase activity in the brain tissue at the border of the missile track established the vitality of the gunshot effect. The presence of macrophages (CD68-epitope) allowed demarcation of a 1-2mm wide necrotic zone around the permanent cavity. Within this zone and beyond, beta-APP showed an initial increase followed by a decline in the number of injured axons. Three types of beta-APP positive staining could be differentiated. In the immediate vicinity of the missile track beta-APP positive neurons were present at a distance of 2-4mm from the margin of the permanent cavity (type 1) as a result of primary injured neuronal tissue by the gunshot itself. At longer distances from the narrow channel and the permanent cavity single beta-APP positive axons or axon fragments and two additional types were found; type 2 shows a parallel, wave-like arrangement of the damaged fibers, which suggests that the injury was produced by mechanical acceleration of the brain tissue created by the energy the projectile expended within the brain; irregular aggregation of beta-APP positive axons or axon fragments within a local edema represents type 3, which may be attributed to secondary ischemia or edema.

[The mitochondrial genome and aging]. / Das mitochondriale Genom und Altern.

There is a lot of evidence that age-associated alterations of the mitochondrial genome occur, especially in postmitotic tissues such as brain, heart and skeletal muscle. These alterations are supposed to be a result of an attack of free radicals generated as normal byproducts of oxidative phosphorylation and lead to damage of proteins, lipids, and DNA. The alterations of mtDNA include oxidative damage of base pairs, point mutations, large-scale deletions or duplications. The 4977 bp deletion or "common deletion" reveals an age-dependent accumulation in postmitotic tissues, but not in fast-dividing tissues such as blood cells. In addition, it is observed that a tissue-specific accumulation occurs with the highest abundance in the basal ganglia, followed by skeletal muscle, heart, and lowest in cerebellar tissue. Third, pathological alterations of specific tissue, like ischemia/reperfusion events, display a pronounced accumulation of the deletion compared to age-matched controls. Because there are many mtDNA mutations, further analysis of all alterations of mtDNA will elucidate its role in the phenomenon of aging. Despite some criticisms of this free radical theory of aging, there is a lot of experimental evidence to support the important role of mitochondria in organismal aging.

Histochemical characterization of cytotoxic brain edema. Potassium concentrations after cerebral ischemia and during the postmortem interval.

Cytotoxic edema is a phenomenon of the ischemically damaged brain. In the present study we tested a histochemical method that detects this phenomenon based on potassium (K+) levels in the brain. In a first series focal cerebral ischemia was induced by arterial occlusion in 23 gerbils (Meriones unguiculatus). After survival times of 30, 60 and 120 min, the animals were killed and brain section histochemically stained for potassium and quantitatively evaluated with a morphometric method. The results were compared with those using physicochemical techniques. A distinct K+ depletion could be demonstrated in the area of the focal ischemia within a survival time of 30 min, the depletion growing thereafter with increasing survival time. In a second series histochemical and chemical methods were used to study the stability of K+ levels in undamaged brains of 15 healthy rats during postmortem intervals of 2.5 and 5 h. Within these intervals K+ levels were clearly depleted, apparently as a result of cerebral spinal fluid (CSF) diffusion. Even if neuronal injury can be demonstrated histochemically after very brief survival times of about 30 min, postmortem storage of the cadavers rendered detection impossible due to electrolyte and water diffusion. In autoptic human cases, therefore, this technique is of no practical utility in detecting cytotoxic brain edema in postmortem tissue.

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.