Effect of the N-methyl-D-aspartate NR2B subunit antagonist ifenprodil on precursor cell proliferation in the hippocampus.

The N-methyl-D-aspartate (NMDA) receptor, one of the ionotropic glutamate receptor, plays important physiological and pathological roles in learning and memory, neuronal development, acute and chronic neurological diseases, and neurogenesis. This work examines the contribution of the NR2B NMDA receptor subunit to adult neurogenesis/cell proliferation under physiological conditions and following an excitotoxic insult. We have previously shown in vitro that a discrete NMDA-induced, excitotoxic injury to the hippocampus results in an increase in neurogenesis within the dentate gyrus. Here we have characterized adult neurogenesis or proliferation, using BrdU, in an in vivo model of excitotoxic injury to the CA1 subfield of the hippocampus. We demonstrate a peak in neural stem cell proliferation/neurogenesis between 6 and 9 days after the excitotoxic insult. Treatment with ifenprodil, an NR2B subunit-specific NMDA receptor antagonist, without prior injury induction, also increased the number of BrdU-positive cells within the DG and posterior periventricle, indicating that ifenprodil itself could modulate the rate of proliferation. Interestingly, though, the increased level of cell proliferation did not change significantly when ifenprodil was administered following an excitotoxic insult. In conclusion, our results suggest and add to the growing evidence that NR2B subunit-containing NMDA receptors play a role in neural stem cell proliferation.

BH3-only proteins BIM and PUMA in the regulation of survival and neuronal differentiation of newly generated cells in the adult mouse hippocampus.

Neurogenesis persists in the adult hippocampus, where several thousand neurons are born every day. Most of the newly generated cells are eliminated by apoptosis, possibly because of their failure to integrate properly into neural networks. The BH3-only proteins Bim and Puma have been shown to mediate trophic factor withdrawal- and anoikis-induced apoptosis in various systems. We therefore determined their impact on proliferation, survival, and differentiation of adult-generated cells in the mouse hippocampus using gene-deficient mice. Wild-type, bim-, and puma-deficient mice showed similar rates of precursor cell proliferation, as evidenced by 5-bromo-2-deoxyuridine (BrdU)-incorporation. Deficiency in either bim or puma significantly increased the survival of adult-born cells in the dentate gyrus (DG) after 7 days. Consistently, we detected increased numbers of doublecortin (DCX)-positive and fewer terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelled-positive cells in the DG of bim- and puma-deficient mice. Bim and puma deficiency did not change early markers of neuronal differentiation, as evidenced by BrdU/DCX double-labelling. However, BrdU/NeuN double-labelling revealed that deficiency of bim, but not puma, accelerated the differentiation of newly generated cells into a neuronal phenotype. Our data show that Bim and Puma are prominently involved in the regulation of neuronal progenitor cell survival in the adult DG, but also suggest that Bim has an additional role in neuronal differentiation of adult-born neural precursor cells.

Active secretion of S100B from astrocytes during metabolic stress.

In patients suffering from cerebrovascular diseases and traumatic brain damage, increases in serum levels of protein S100B are positively correlated with the severity of the insult. Since high concentrations of S100B have been shown to exert neurotoxic effects, the objective of this study was to characterize the regulatory mechanisms underlying control of S100B release from astrocytes. To that end, we analyzed the kinetics and amount of S100B release in correlation with regulation of S100B gene expression in an in vitro ischemia model. Astrocyte cultures were treated with combined oxygen, serum and glucose deprivation, serum and glucose deprivation or hypoxia alone for 6, 12 and 24 h, respectively. While oxygen, serum and glucose deprivation triggered the most rapid release of S100B, serum and glucose deprivation provoked comparable levels of released S100B at the later time points. In contrast to oxygen, serum and glucose deprivation and serum and glucose deprivation, hypoxia alone elicited only marginal increases in secreted S100B. Parallel analysis of extracellular lactate dehydrogenase and the number of viable cells revealed only moderate cell death in the cultures, indicating that S100B was actively secreted during in vitro ischemia. Interestingly, S100B mRNA expression was potently downregulated after 12 and 24 h of oxygen, serum and glucose deprivation, and prolonged oxygen, serum and glucose deprivation for 48 h was associated with a significant reduction of S100B release at later time intervals, whereas lactate dehydrogenase levels remained constant. Our data suggest that secretion of S100B during the glial response to metabolic injury is an early and active process.

Molecular interactions of the type 1 human immunodeficiency virus transregulatory protein Tat with N-methyl-d-aspartate receptor subunits.

We investigated the effect of type 1 human immunodeficiency virus (HIV-1) regulatory protein Tat on N-methyl-d-aspartate (NMDA) receptors expressed in Xenopus oocytes by voltage-clamp recording and its role in NMDA-mediated neurotoxicity using cultured rat hippocampal neurons. Tat (0.01-1muM) potentiated NMDA-induced currents of recombinant NMDA receptors. However, in the presence of Zn(2+), the potentiating effect of Tat was much more pronounced, indicating an additional Zn(2+)-related effect on NMDA receptors. Consistently, Tat potentiated currents of the particularly Zn(2+)-sensitive NR1/NR2A NMDA receptor with a higher efficacy, whereas currents from a Zn(2+)-insensitive mutant were only marginally augmented. In addition, chemical-modified Tat, deficient for metal binding, did not reverse Zn(2+)-mediated inhibition of NMDA responses, demonstrating that Tat disinhibits NMDA receptors from Zn(2+)-mediated antagonism by complexing the cation. We therefore investigated the interplay of Tat and Zn(2+) in NMDA-mediated neurotoxicity using cultures of rat hippocampal neurons. Zn(2+) exhibited a prominent rescuing effect when added together with the excitotoxicant NMDA, which could be reverted by the Zn(2+)-chelator tricine. Similar to tricine, Tat enhanced NMDA-mediated neurotoxicity in the presence of neuroprotective Zn(2+) concentrations. Double-staining with antibodies against Tat and the NR1 subunit of the NMDA receptor revealed partial colocalization of the immunoreactivities in membrane patches of hippocampal neurons, supporting the idea of a direct interplay between Tat and glutamatergic transmission. We therefore propose that release of Zn(2+)-mediated inhibition of NMDA receptors by HIV-1 Tat contributes to the neurotoxic effect of glutamate and may participate in the pathogenesis of AIDS-associated dementia.

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.

S100B potently activates p65/c-Rel transcriptional complexes in hippocampal neurons: Clinical implications for the role of S100B in excitotoxic brain injury.

Increased serum levels of S100B are positively correlated with multiple forms of CNS damage, such as stroke, CNS trauma and neurodegenerative diseases, but also in psychiatric disorders. However, it is currently not known whether increased serum levels of S100B reflect a neuroregenerative or neurodegenerative response. Since glutamate receptor overactivation (excitotoxicity) may contribute to neuronal pathology in psychiatric disorders, we investigated the effect of S100B on N-methyl-d-aspartate (NMDA)-induced neuronal cell death. Here we demonstrate that very low concentrations of S100B significantly protect primary rat hippocampal neurons against NMDA toxicity by activation of transcription factors of the Rel/nuclear factor kappaB (NF-kappaB) family. Further experiments suggest that i) S100B activated expression of the receptor of advanced glycation products (RAGE) gene in neurons and ii) S100B induced a unique composition of the active NF-kappaB complex consisting of the p65 and c-Rel subunits suggesting a novel mechanism for NF-kappaB activation involved in S100B-mediated neuroprotection. Our data suggest that S100B secreted during the glial response to brain injury potently activates p65/c-Rel in a RAGE-dependent manner and may exert neuroprotective and neuroregenerative effects in psychiatric disorders.

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.

Fatal pedestrian-bicycle collisions.

Although, fatal collisions between pedestrians and bicycles are relatively rare, they are still of forensic relevance because of the need to explore the circumstances of the accident. Based on three reconstructed cases, situation and injury patterns are presented that might prove useful in future cases: usually the person causing the accident is the cyclist while the pedestrian generally suffers more severe injuries; the situation at the site of accident is important for its reconstruction: end location of the persons involved in the accident, injuries and traces on pedestrians and cyclists, traces at the site of accident and on the bicycle; because of the lack of pre-crash traces and any eyewitness accounts, the pedestrian's injuries are the best starting point for the reconstruction of the accident; a characteristic wound on the lower leg of the pedestrian that reveals the initial impact between the front wheel and the leg is crucial not because of its seriousness, but because of its external morphology; the injuries that can be expected by the following impact between body and handlebar are unspecific and only minor; the most severe injuries to the pedestrian as a result of the accident are caused secondarily by falling and hitting the head on the road; the fall of the cyclist, however, corresponds to a throw-off followed by a sliding phase with less impact load when the head hits the ground [maximum abbreviated injury scale 1 (MAIS 1)]; the cyclists involved are mainly younger persons on fashionable bicycles (here: mountain bikes); in the great majority of cases, the injured pedestrians are frail, elderly people with a lower tolerance of trauma.

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.

Brain injury after gunshot wounding: morphometric analysis of cell destruction caused by temporary cavitation.

In addition to the primary destruction of brain tissue readily visible at autopsy (permanent track), gunshot wounding to the brain creates a pulsating temporary cavity due to radial expansion along the bullet's track. To determine the maximum extent of this temporary cavitation in brains of victims of gunshots from weapons with low muzzle velocity, we carried out morphometric studies on 20 cases of death from gunshot wounding to the head from bullets with a muzzle energy <500 J and a survival time of <90 min. The brains were fixed in formalin, examine macroscopically and microscopically, and subjected to morphometric analyses. Surrounding the permanent track, a narrow mantle-like zone of astrocyte destruction was found within an area of hemorrhagic extravasation. Axons near the permanent track had been broken into tiny fragments. The axonal damage lessened with increasing distance from the permanent track, although axons continued to be fragmented and to exhibit varicose changes and clumping until assuming their normal structure beyond 18 mm. Nerve cells were extremely shrunken close to the permanent track but gradually took on their normal shape with increasing distance. We also assessed the loss of glial fibrillary acid protein expression by astrocytes in the white matter, the extent of traumatic bleeding, and damage to axons and neurons as measured radially from the permanent track. Axonal and neuronal damage were found to extend about 18 mm radially from the permanent track, tapering gradually along the track from entry point to exit point. The destruction was probably produced by the temporary cavitation and accords with theoretical considerations and experimental observations.

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

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 beta-amyloid precursor protein (beta-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 beta-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.

Axonal injury--a diagnostic tool in forensic neuropathology? A review.

We used beta-amyloid precursor protein (beta-APP) to investigate our own forensic neuropathological case material (n = 252) in light of the current literature on the phenomenon "axonal injury" (AI) to determine the incidence, specificity and biomechanical significance of AI and its significance for determining vitality and survival time. The case material consisted of cases of fatal nonmissile closed-head injury (n = 119), gunshot injury (n = 30), fatal cerebral ischemia/hypoxia (n = 51), brain death caused by mechanical trauma (n = 14) or nonmechanical injury (n = 18), and acute hemorrhagic shock (n = 20). AI was observed in 65% to 100% of cases of closed-head injury, fatal cerebral ischemia/hypoxia, and brain death with a survival time of more than 3 h; AI could not be detected in the cases of acute hemorrhagic shock. A statistically significant difference between traumatically and nontraumatically induced (nondisruptive) AI was not found. There was no statistical evidence of a correlation between AI and the different types of external force, since AI could be demonstrated after both acceleration/deceleration injuries and traumatic impact. Therefore, biomechanical inferences for reconstruction purposes are not possible. On the other hand, beta-APP was found to be a definite marker of vitality. In our material, cases with a posttraumatic interval of under 180 min did not express beta-APP. Moreover, the literature shows that the posttraumatic interval can be determined by other methods for demonstration of AI such as by ubiquitin immunostaining (360 min), silver staining (15-18 h), hematoxylin and eosin staining (about 24 h), or by demonstration of a microglial reaction (about 4 to 10 days) or of a few remaining isolated bulbs, without accompanying fibers, which can be detected after a survival time of up to 17 months.

[Observations of the propagation velocity and formation mechanism of burst fractures caused by gunshot]. / Beobachtungen zur Ausbreitungsgeschwindigkeit und Entstehungsursache von Berstungsfrakturen beim Schuss.

In ballistic literature bursting fractures of the skull in gunshot wounds of the head are understood to be produced by the high pressure built up in the cranial cavity as a result of temporary cavity formation. On one hand there is known, that maximum expansion of the temporary cavity does not occur until some time after the bullet has passed through the skull. On the other hand there exist observations that bursting fractures originating from entrance hole traversed the skull more rapidly than did the projectile. In this paper there is presented a case with such a phenomenon in humerus. Since the determined fracture propagation velocity is very high, there is postulated a direct formation mechanism preceding the indirect effect of the temporary cavity. This hypothesis is confirmed by experiment.

[Criteria for differentiating self- or other-inflicted injuries of alleged or real victims]. / Unterscheidungskriterien für Selbst- oder Fremdbeibringung von Verletzungen angeblich oder tatsächlich Uberfallener.

A synoptic study of six cases of self-inflicted sharp force injuries is presented, where young, mostly female people had simulated assaults to gain sympathy or other personal advantage. The morphological distinctives of simulated assault wounds from such of real assault are worked out and the common situative and motivative background is cleared up.

[Cytologic findings as indications: morphologic and immunohistochemical identification of human brain tissue on the clothing of a murderer]. / Zytologischer Befund als Indiz: Morphologische und immunhistochemische Identifizierung von menschlichem Hirngewebe an der Täterkleidung.

A case was described in which a murder was concealed by burning the body. The suspect confessed to committing the crime after being informed that human brain tissue in form of slimy stains as well the same blood group as the victim had been identified on his clothing. Slimy stains (approx. 2 x 3 mm) were identified as brain tissue by sectioning on the cryostat together with the bits of wool fiber. Morphologically, nerve cells were clearly identifiable, particular by demonstrating Nissl's bodies. Application of anti-human serum protein and an anti-human cerebrum serum confirmed this finding and augmented it by confirming that the brain tissue was of human origin. The unusual character of this case and the methods used prompted the authors to publish these findings. The available literature is discussed.

[Incidents and hazards in the use of blank metal clamps with venous catheters]. / Zwischenfälle und Gefahren bei der Verwendung nicht armierter Metallklemmen an Venenkathetern.

The use of blank metal clamps resulted in severe damaging of infusion systems in two cases. In one case the silicone tube of a Shaldon catheter was damaged by the clamps and thus became detached from the Luer-Lock syringe connecting piece, whereupon the patient bled to death. In another case the too forcible screwing while fastening a Luer-Lock coupling by means of a metal clamp produced a tear in the connecting piece. The female patient, who sat upright, collapsed and died under the clinical suspicion of air embolism, which, however, could not be confirmed at autopsy. Such incidents are avoidable only if doctors and nurses are carefully trained in the handling of vascular catheters and infusion systems and if they are expressly informed not to use blank metal clamps.

[Intravital and postmortal CT examinations in cerebral gunshot injuries]. / Intravitale und postmortale CT-Untersuchungen bei Hirnschussverletzungen.

The value of CT was assessed in 24 patients who died of cerebral gunshot injuries and in two patients with more recent injuries in order to reconstruct the mode of injury and for adding forensic information. The post-mortem and intravital appearances described and are compared with ultrasound rotation compound scans of the isolated brains. CT showed good agreement with pathological findings. Ultrasound produced images with an accuracy between CT and photographs of the brain specimen. Both methods are regarded as valuable additions to the pathological and forensic information concerning gunshot injuries.