Bernd Schmitz

Activation of CPP-32 protease in hippocampal neurons following ischemia and epilepsy

Recent in vitro studies indicate an involvement of members of the interleukin-1beta converting enzyme (ICE) family of proteases in programmed neuronal cell death. Cell death of hippocampal neurons in animal models of cerebral ischemia and epilepsy shows morphological features of apoptosis and can be prevented by administration of protein synthesis inhibitors suggesting that de novo synthesis of components of the cell death program is necessary for neuronal apoptosis. In the present study we demonstrate by in situ hybridization analysis that expression of CPP-32, an ICE-related protease, is significantly upregulated in CA1 hippocampal neurons following global ischemia induced by cardiac arrest and in hippocampal neurons of the CA3/CA4 region after kainate-mediated epilepsy, respectively. Moreover, an increase in CPP-32-like proteolytic activity was detected in hippocampal extracts 24 h after ischemia using the fluorogenic CPP-32 substrate Ac-DEVD-AMC. Activation of CPP-32 clearly preceded cell death of hippocampal neurons as assessed by in situ end-labelling of nuclear DNA fragments. These results indicate that CPP-32 protease may be activated at both the transcriptional and post-translational level during neuronal apoptosis and that activation correlates with the selective vulnerability of hippocampal pyramidal neurons to ischemic and epileptic insults.

Effect of global system for mobile communication (GSM) microwave exposure on blood-brain barrier permeability in rat

Abstract We investigated the effects of global system for mobile communication (GSM) microwave exposure on the permeability of the blood-brain barrier using a calibrated microwave exposure system in the 900 MHz band. Rats were restrained in a carousel of circularly arranged plastic tubes and sham-exposed or microwave irradiated for a duration of 4 h at specific brain absorption rates (SAR) ranging from 0.3 to 7.5 W/kg. The extravasation of proteins was assessed either at the end of exposure or 7 days later in three to five coronal brain slices by immunohistochemical staining of serum albumin. As a positive control two rats were subjected to cold injury. In the brains of freely moving control rats (n = 20) only one spot of extravasated serum albumin could be detected in one animal. In the sham-exposed control group (n = 20) three animals exhibited a total of 4 extravasations. In animals irradiated for 4 h at SAR of 0.3, 1.5 and 7.5 W/kg (n = 20 in each group) five out of the ten animals of each group killed at the end of the exposure showed 7, 6 and 14 extravasations, respectively. In the ten animals of each group killed 7 days after exposure, the total number of extravasations was 2, 0 and 1, respectively. The increase in serum albumin extravasations after microwave exposure reached significance only in the group exposed to the highest SAR of 7.5 W/kg but not at the lower intensities. Histological injury was not observed in any of the examined brains. Compared to other pathological conditions with increased blood-brain barrier permeability such as cold injury, the here observed serum albumin extravasations are very modest and, moreover, reversible. Microwave exposure in the frequency and intensity range of mobile telephony is unlikely to produce pathologically significant changes of the blood-brain barrier permeability.

Subjective memory complaints: Objective neural markers in patients with Alzheimer's disease and major depressive disorder

Patients with probable Alzheimers disease and depressive patients frequently present with subjective memory complaints. Objective distinction of underlying neuronal substrate malfunction and early cross‐sectional differential diagnosis have been elusive thus far. We used repetitive learning and free recall of abstract geometric patterns during functional magnetic resonance imaging to assess episodic memory in older subjects (ages 56–64 years) who sought first‐time medical attention with subjective memory complaints and were diagnosed with probable Alzheimers disease (NINCDS‐ADRDA criteria; ages 51–67 years) or major depressive disorder (DSM‐IV; ages 50–65 years). Contrasting healthy seniors or depressive patients with Alzheimers disease patients revealed superiority of hippocampal activation. Contrasting Alzheimers disease patients with seniors showed bilateral prefrontal activity as a correlate of futile compensation of episodic memory failure. Contrasting patients who had major depressive disorder with seniors or patients who had Alzheimers disease showed bilateral activation of the orbitofrontal cortex and the anterior cingulate. Subjective memory complaints may be classified objectively and very early with functional magnetic resonance imaging of episodic memory in groups of patients with Alzheimers disease and depressive syndrome. This may facilitate drug trials with evaluation of specific treatments, but further studies will be needed to establish the differential diagnosis for the individual patient.

Neuronal Stress Response and Neuronal Cell Damage After Cardiocirculatory Arrest in Rats

Cardiocirculatory arrest is the most common clinical cause of global cerebral ischemia. We studied neuronal cell damage and neuronal stress response after cardiocirculatory arrest and subsequent cardiopulmonary resuscitation in rats. The temporospatial cellular reactions were assessed by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick endlabeling (TUNEL) staining of DNA fragments, in situ hybridization (heat shock protein hsp70; immediate early genes c-fos and c-jun), and immunocytochemical (HSP70; and myeloperoxidase, specific marker of polymorphonuclear leukocytes [PMNL]) techniques. Cardiac arrest of 10 minutes duration was induced in mechanically ventilated male Sprague-Dawley rats anesthetized with nitrous oxide and halothane. After cardiopulmonary resuscitation, animals were allowed to reperfuse spontaneously for 6 hours, 24 hours, 3 days, and 7 days (n = 6 per group). Five sham-operated animals were controls. The TUNEL staining revealed an early onset degeneration in the thalamic reticular nucleus (TRN) at 6 hours that peaked at 3 days. In contrast, degeneration was delayed in the hippocampal CA1 sector, showing an onset at 3 days and a further increase in the number of TUNEL-positive cells at 7 days. A minor portion of TUNEL-positive nuclei in the CA1 sector showed condensed chromatin and apoptotic bodies, whereas all nuclei in the TRN revealed more diffuse staining. After 6 hours of reperfusion, levels of mRNA for hsp70 and c-jun were elevated in circumscribed areas of cortex, in all hippocampal areas, and in most nuclei of thalamus, but not in the TRN. After 24 hours, a strong expression of mRNA for hsp70 and c-jun could be observed in the second layer of the cortex and in hippocampal CA1 sector; hsp70 also was observed in hippocampal CA3 sector. Some animals showed expression of hsp70 and c-jun in the dentate gyrus. After 3 days, hsp70 and c-jun were detected mainly in the CA1 sector of hippocampus. At 7 days, mRNA for both returned to control values. Therefore, delayed cell degeneration in the CA1 sector corresponds to a prolonged expression of hsp70 and c-jun in this area. In situ hybridization studies for c-fos revealed a strong signal in CA3 and dentate gyrus and a less prominent signal in TRN at 6 hours. At 24 hours, CA4 and amygdalae were positive, whereas at 3 and 7 days, the signal reached control levels; no prolonged or secondary expression was observed in the CA1 sector. Immunohistochemical study confirmed translation of HSP70 in various areas corresponding to the detection of mRNA, including the CA1 sector. The number of PMNL increased significantly at 6 hours and 7 days after cardiac arrest; PMNL were distributed disseminately and were not regionally associated with neuronal cell damage. The current data support the view that CA1 neurons might undergo an apoptosis-associated death after cardiac arrest, but PMNL are not directly involved in this process. The marked differences in the time course and the characteristics of TUNEL staining and the neuronal stress response in CA1 sector and TRN point to different mechanisms of neuronal injury in the two selectively vulnerable areas.

The cerebral 'no-reflow' phenomenon after cardiac arrest in rats--influence of low-flow reperfusion.

OBJECTIVEnExperimental data indicate that early microcirculatory reperfusion is disturbed after cardiac arrest. We investigated the influence of prolonged cardiac arrest and basic life support (BLS) procedures on the quality of cerebral microcirculatory reperfusion.nnnMATERIALS AND METHODSnIn mechanically ventilated male Wistar rats anesthetized with N2O and halothane, cardiac arrest was induced by electrical fibrillation. Ten animals (group I) were subjected to 17 min of cardiac arrest (no-flow). Nine additional animals (group II) underwent only 12 min of cardiac arrest (no-flow), which was followed by a 5-min phase of BLS (i.e. mechanical ventilation and external cardiac compressions). In both groups, advanced resuscitation procedures including mechanical ventilation, external cardiac massage, 0.2 mg kg-1 epinephrine, 0.5 mmol kg-1 NaHCO3, and defibrillation were started 17 min after induction of cardiac arrest. The perfusion of the cerebral microcirculation was visualized by injection of 0.3 g kg-1 15% fluorescein isothiocyanate (FITC)-albumin 5 min after restoration of spontaneous circulation (ROSC), and the animals were decapitated 2 min later. The left hemispheres were fixed in 4% formalin, and coronal sections of 200 microns thickness at three different standard levels of the rat brain were investigated using fluorescence microscopy. Areas without capillary filling (cerebral no-reflow) were identified and calculated.nnnRESULTSnROSC could be achieved in five of 10 animals (50%) of group I, and in six of nine animals (67%) of group II (P = n.s.). The severity of cerebral no-reflow was higher in group II compared with group I (6.9 +/- 7.6 vs. 0.7 +/- 0.7% of total sectional areas; P < or = 0.05). Two sham-operated animals showed homogeneous reperfusion.nnnCONCLUSIONSnWistar rats did not develop a marked cerebral no-reflow phenomenon after circulatory arrest. A relevant degree of cerebral no-reflow occurred, however, in animals subjected to a phase of BLS before circulatory stabilization. Therefore, low-flow states following prolonged cardiocirculatory arrest may aggravate early cerebral microcirculatory reperfusion disorders.

Ultrafast Perfusion-Weighted MRI of Functional Brain Activation in Rats During Forepaw Stimulation: Comparison with T*2-Weighted MRI

A fast version of the arterial spin tagging technique for the detection of cerebral perfusion is presented. Based on adiabatic spin inversion in combination with snapshot FLASH imaging, our technique allows the recording of perfusion changes with a temporal resolution of about 3u2009s. Differences of cerebral perfusion dependent on the choice of anesthesia were observed in rat brain. Furthermore, with this arterial spin tagging method we demonstrated perfusion increases in the somatosensory cortex of anaesthetized rats during forepaw stimulation. Comparison of the activated areas in the T*2‐weighted BOLD images and the perfusion‐weighted images showed good spatial correspondence, but the sensitivity to the functional activation was more than ten times higher in the perfusion technique.

Penumbral tissue alkalosis in focal cerebral ischemia: relationship to energy metabolism, blood flow, and steady potential.

The effect of focal ischemia on tissue pH was studied at various times up to 6 hours after permanent middle cerebral artery occlusion in rats. Tissue pH was imaged by using umbelliferone fluorescence and correlated with cerebral blood flow, ATP content, and recordings of the steady potential. Circumscribed foci of alkalosis (pH 7.32 ± 0.11) were detected with increasing frequency in penumbral regions having near‐to‐normal ATP concentrations and cerebral blood flow values between 20% and 40% of control. Both the infarct core, defined by ATP loss and cerebral blood flow values of less than 20% of control, and the inner peri‐infarct rim were consistently acidic (pH 6.03 ± 0.36 and 6.53 ± 0.24, respectively). Treatment with the glutamate antagonist dizocilpine (MK‐801) suppressed negative shifts of the steady potential and reduced significantly the occurrence of alkalosis observed in 90% of untreated but only in 44% of treated animals. Penumbral alkalosis appeared to be a time‐dependent event occurring 30 to 60 minutes after the passage of peri‐infarct depolarizations. The diversity of penumbral pH changes reflects the local disturbance of pH regulation and, possibly, the differential fate of penumbral subareas. Ann Neurol 2000;47:485–492.

Regional and directional anisotropy of apparent diffusion coefficient in rat brain

Quantitative diffusion maps were recorded in normal rat brain. In multi‐slice sections covering the whole brain, strong variation of the apparent diffusion coefficient (ADC) was observed depending on slice position at constant gradient direction. Furthermore, a varying difference between apparent diffusion coefficients depending on gradient direction was found, reaching 32% in the cortex of the ventral‐most horizontal sections while showing equal ADC on the dorsal cortex side. The regional variation and directional anisotropy of the ADC was not restricted to white matter but was described for both cortical and subcortical brain tissue. From diffusion coefficients along the three major field gradient directions (ADC x , ADC y , ADC z ), the average ADC (ADCaverage) was determined from the trace of the diffusion tensor (D) as 653u2005±u200528 µm2/s for parietal cortex and 671u2005±u200532 µm2/s for lateral cortex, independent of position along the sagittal direction. From these observations about the regional diffusion anisotropy, a more stringent protocol for the description of ischemic ADC changes is proposed. Copyright

Functional activation of cerebral blood flow after cardiac arrest in rat.

After a period of global cerebral ischemia, CO2 reactivity and the hemodynamic–metabolic activation to functional stimulation are transiently suppressed. This raises the question of whether the impaired functional coupling reflects disturbances of functional integrity of the brain or an impaired cerebrovascular reactivity. We, therefore, compared the recovery of CO2 reactivity with that of somatosensory evoked potentials, functional flow activation and neurologic deficits in a rodent model of cardiac arrest-induced cerebral ischemia, followed by up to 7 days of reperfusion. Cardiac arrest of 10 minutes duration was produced in 24 animals by electrical fibrillation of the heart. Five animals were sham-operated controls. Resuscitation was performed by external cardiac massage, using standard resuscitation procedures. Functional activation was carried out under chloralose anesthesia by electrical stimulation of forepaws. CO2 reactivity was tested by ventilation of animals with 6% CO2. During functional and hypercapnic stimulation CBF was measured in the somatosensory cortex using laser–Doppler flowmetry, and at the end of the experiment by 14C-iodoantipyrine autoradiography. Neurologic deficits were scored by evaluating consciousness and various sensory and motor functions. In control animals 6% CO2 increased CBF measured by laser–Doppler flowmetry by 28.8% ± 8.7%. Forepaw stimulation generated somatosensory evoked potentials with an amplitude of 750 ± 217 μV and increased CBF measured by laser–Doppler flowmetry by 86.0% ± 18.1%. After return of spontaneous circulation, CO2 reactivity was transiently reduced to about 30% of control at 1 hour of reperfusion (P < 0.05) but returned to near control at 5 hours. Somatosensory evoked potential amplitudes were reduced to 15% of control at 45 minutes of reperfusion and returned to only 50% to 60% at 3 and 7 days after return of spontaneous circulation (P < 0.05). Functional activation of blood flow was completely suppressed during the first hour after return of spontaneous circulation but also recovered to 50% to 60% of control at 3 days after return of spontaneous circulation (P < 0.05). Linear regression analysis revealed a significant correlation between recovery of functional activation of blood flow and both recovery of the amplitude of somatosensory evoked potentials (P = 0.03) and the neurologic deficit score (P = 0.02), but not between neurologic deficit score and recovery of CO2 reactivity or somatosensory evoked potential amplitudes. These data demonstrate that the suppression of functional activation of blood flow after 10 minutes cardiac arrest is not related to impairment of coupling mechanisms but reflects ongoing disturbances of the functional integrity of the brain. Assessment of functional flow coupling is a reliable way to study postischemic recovery of the brain.

Imaging of the apparent diffusion coefficient for the evaluation of cerebral metabolic recovery after cardiac arrest

The apparent diffusion coefficient (ADC) of water is a sensitive indicator of water and ion homeostasis of brain. Resuscitation of the brain after cardiac arrest, the most frequent reason for global cerebral ischemia under clinical conditions, depends critically on the reversal of disturbances of water and ion homeostasis. We, therefore, investigated whether ADC imaging can be used to monitor the development and reversal of ischemic brain injury during and after cardiac arrest. Ten adult mongrel normothermic cats were anesthetized with alfentanil and midazolam, immobilized with pancuronium, and mechanically ventilated with O2/N2O. Arterial, left ventricular, central venous, and intracranial pressures were monitored throughout the experiment. Magnetic resonance imaging was performed in a 4.7T MR scanner with a shielded gradient system. Diffusion-weighted images (DWI) were obtained by pulsed gradient spin echoes (diffusion weighting factor b: 0, 500, 1000, 1500 s/mm2). Quantitative ADC images were calculated from DWIs by fitting signal intensities against b-factors. Fifteen minute cardiac arrest was induced in the magnet by electrical fibrillation. Resuscitation was also carried out in the magnet, using a pneumatic vest for remotely controlled closed chest cardiac massage. Seven of 10 animals were resuscitated successfully and subsequently monitored for 3 h. During cardiac arrest, ADC declined from 678 +/- 79 x 10(-6) to 430 +/- 128 x 10(-6) mm2/s (63% of baseline). In the successfully resuscitated animals ADC returned to 648 +/- 108 x 10(-6) mm2/s within 30 min and remained at this level throughout the 3 h of recirculation. Regional evaluations of ADC revealed a transient overshoot in brainstem and basal ganglia to 114% of control at 15 min before returning to baseline values after 40 min. Failure of cardiac resuscitation prevented ADC normalization and led to its further decline to below 50% of control. Postcardiac arrest normalization of ADC maps correlated with homogeneous return of ATP, glucose, and lactate to near normal, whereas failure of ADC normalization was associated with depletion of ATP and glucose and severe lactate accumulation. In conclusion, our data indicate, that normalization of ADC is a reliable indicator of cerebral recovery after resuscitation from cardiac arrest.