M. Oehmichen

Neuropathology in non-human immunodeficiency virus-infected drug addicts: hypoxic brain damage after chronic intravenous drug abuse

Abstract Neuropathological studies were carried out on 180 human immunodeficiency virus-seronegative intravenous drug addicts. The findings in victims of acute heroin intoxication (n = 116) were congestion (99.1%), capillary engorgement (68.1%), and/or perivascular bleeding (68.1%) – hemodynamic processes attributable to toxic primary respiratory failure. In a high percentage of these cases (88%), cerebral edema was also present. In 18 cases of acute heroin intoxication who survived for periods of hours or days, the sole postmortem finding was ischemic nerve cell damage, resembling that typically seen in systemic hypoxia. Semiquantitative analysis revealed nerve cell loss in the hippocampal formation and/ or Purkinje cell layer in 26% of the 162 chronic drug abusers. By contrast, in nearly 80% of these cases, the hippocampus showed enhanced expression of glial fibrillary acid protein by astrocytes and/or a proliferation of microglia, demonstrated by CD68 expression. Since such reactive processes are produced by primary neuronal damage, it can be assumed that chronic intravenous drug abuse results in obviously ischemic nerve cell loss. This could be demonstrated in the hippocampus, but it must also occur throughout the whole brain. The demonstration of ischemic nerve cell damage and neuronal loss or secondary reactive alterations has not been described previously.

Is traumatic axonal injury (AI) associated with an early microglial activation? Application of a double-labeling technique for simultaneous detection of microglia and AI

Abstract The aim of the present study was to determine whether axonal injury (AI) induces a microglial reaction within 15 days after brain trauma. In 40 selected cases of confirmed AI, the topographical relation of AI and microglial reaction was assessed using an immunohistochemical double-labeling technique for simultaneous demonstration of AI using β-amyloid precursor protein (β-APP) antibody and of microglia using CD68 antibody. Although traumatic injury was usually followed by a moderate early diffuse rise in the number of CD68-reactive cells in the white matter, increases in macrophages in areas of AI accumulation were only sporadic and did not occur until after 4 days. At survival intervals of 5–15 days a moderate microglial reaction in regions of β-APP-positive injured axons was detected, at maximum, in half of the case material. During this interval AI-associated satellitosis-like clusters or stars described by other authors after a survival time of more than 7 weeks were an isolated phenomenon. The prolonged microglial reaction as well as the reduction of β-APP-positive AI during longer survival periods supports the hypothesis that AI is not primarily chemotactically attractive and that the damage to a portion of β-APP-stained axons may be partly reversible. Most cases clearly require a prolonged interval of more than 15 days before initiation of the final scavenger reaction. For forensic purposes the increase in the number of microglial cells within the region of AI accumulation after a survival time of more than 5 days and the multiple and distinct demonstration of star-like microglial reactions within the white matter after survival times exceeding 7 weeks may provide valuable postmortem information on the timing of a traumatic event.

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.

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.

Pontine axonal injury after brain trauma and nontraumatic hypoxic-ischemic brain damage.

Abstract Experimental studies have shown that diffuse axonal injury is usually induced by positive or negative acceleration mechanisms. In order to determine the reliability of axonal injury (AI) as a marker of this type of traumatic insult, we compared cases of trauma-induced focal cortical hemorrhage without dural involvement (n = 67) with cases of trauma-induced subdural bleeding without cortical hemorrhage (n = 26). Both groups exhibited a wide range of post-traumatic survival times. The injuries in the first group were caused mainly by direct impact to the head, those in the second by acceleration/deceleration mechanisms. The investigations were based primarily on immunohistochemical demonstration of antibodies targeted to β-amyloid precursor protein (β-APP) in the pons as a marker of AI and the results were assessed semiquantitatively. No significant differences were found between the two groups. In both groups AI was detected in 80–100% of cases with survival times of more than 3 h and two thirds of all positive cases showed pronounced positivity. Additional comparison of cases of brain death due to mechanical trauma (n = 14) with cases of brain death due to non-mechanical trauma (n = 18) also disclosed no significant intergroup differences. Finally, investigations of the pons in cases of non-traumatic death due to cerebral hypoxia/ischemia (n = 51) demonstrated AI with the same frequency as in the other groups, although the expression tended to be less pronounced. Our results confirm that β-APP expression in the pons is a reliable indicator of AI but does not discriminate between injuries caused by traumatic strain or shearing mechanisms and secondary damage due to cerebral hypoxia/ ischemia or edema. In the large majority of cases with prolonged post-traumatic survival, it can therefore be assumed that AI in the pons is the consequence of primary and/or secondary events or a combination of both, as is common in non-missile head injury survived for more than 90–120 min. Therefore, positive differentiation of the type of biomechanical event based on this criterion alone is not possible.

The shaking trauma in infants - kinetic chains.

The findings in three children who died as a consequence of shaking and those in another child who survived are presented. In the three fatal cases, a combination of anatomical lesions were identified at autopsy which appear to indicate the sites where kinetic energy related to the shaking episodes had been applied thus enabling the sequence of events resulting in the fatal head injury to be elucidated. Such patterns of injuries involved the upper limb, the shoulder, the brachial nerve plexus and the muscles close to the scapula; hemorrhages were present at the insertions of the sternocleidomastoid muscles due to hyperextension trauma (the so-called periosteal sign) and in the transition zone between the cervical and thoracic spine and extradural hematomas. Characteristic lesions due to traction were also found in the legs. All three children with lethal shaking trauma died from a subdural hematoma only a few hours after the event. The surviving child had persistant hypoxic damage of the brain following on massive cerebral edema. All the children showed a discrepancy between the lack of identifiable external lesions and severe internal ones.

Enhanced reactivity of Alz-50 antibody in brains of sudden infant death syndrome victims versus brains with lethal hypoxic/ischemic injury. Diagnostic significance after application of the ImmunoMax technique on routine paraffin material.

Abstract Alz-50 antibody is immunoreactive with brain tissue of subjects with Alzheimer’s disease and can also be demonstrated by immunocytochemistry in neurons of vibratome-prepared brain tissue of victims of sudden infant death syndrome (SIDS). The application of a slightly modified ImmunoMax method enabled us to demonstrate Alz-50 immunoreactivity in paraffin-embedded material. The Alz-50 epitope was detected in the hippocampus region and in nuclei of the medulla oblongata at the level of the inferior olivary protuberance in three diagnostic groups: victims of SIDS (n = 10), infants dying of subacute hypoxia/ischemia with subsequent (re-)perfusion (n=9), and infants dying of acute ischemia without (re-) perfusion (n = 7). Quantitative evaluation of the hippocampal cortex and the nucleus olivaris inferior disclosed a significantly (P < 0.05) higher percentage of Alz-50-reactive neurons in SIDS cases than in the control groups (hippocampal cortex and nucleus olivaris; SIDS victims: median = 100%; subacute hypoxia/ischemia: median = 33.6– 81%; acute ischemia: median = 89.2–99%). Semiquantitative analysis revealed an equally pronounced preponderance of Alz-50-reactive neurons in SIDS victims versus the control groups. This greater expression in SIDS victims may be due to an ongoing hypoxia/ischemia during agony, but the present paucity of knowledge prohibits definitive elucidation. Nevertheless, the method described here appears to offer the realistic possibility of distinguishing SIDS cases from cases of sudden death in infants due to other causes, i.e., it offers for the first time a positive criterion for the diagnosis of SIDS.

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.

AgNORs during the process of wound healing

Abstract Silver staining of paraffin sections to detect changes in nucleolar organizer regions (AgNORs) is an established method for detecting cellular proliferation. To determine whether AgNORs are helpful in assessing wound age and vitality, we examined intravital and postmortem skin biopsies from rats surviving incised wounds to both pinnae for defined intervals up to 384 h using, 7 rats per time interval. One biopsy was taken immediately before death, a second 24 h after been sacrificed and storage at 8 °C. Interactive computer-assisted image analysis revealed that in the first 120 h after trauma the total number of AgNORs, the mean AgNOR sum area in the nucleus, and the mean AgNOR area per nucleus were dependent on survival time. Taken as indicators of proliferative activity these morphological phenomena revealed an increase in proliferative activity after survival times ranging from 10 to a maximum of 96 h. The findings were the same in both intravital and postmortem biopsies. These findings are in accordance with those obtained using bromodeoxyuridine. The value of these experimental findings in assessing wound age of human beings is discussed.

Diagnostic significance of myofibrillar degeneration of cardiocytes in forensic pathology

The incidence of myofibrillar degeneration (MFD) was studied in the following different forensic-pathological diagnostic groups of 25 cases each: acute morphine intoxication, acute carbon monoxide intoxication, hanging, strangulation by hand/ligature, drowning, acute hemorrhagic shock, lethal acute brain injury, explainable death of babies or infants and sudden infant death syndrome, together with 18 cases of intoxication with various drugs. The MFD was demonstrated by the Luxol-fast-blue reaction, with two types of phenomena being differentiated, namely cross-band lesions and diffuse staining. All diagnostic groups included cases of MFD of differing degrees. Cross-band lesions were observed in practically all cases of hanging, strangulation and acute hemorrhagic shock. Diffuse stain was noted particularly in cases of drowning and acute brain injury. The diagnostic significance is discussed.