K.-A. Hossmann

Repeated negative DC deflections in rat cortex following middle cerebral artery occlusion are abolished by MK-801 : effect on volume of ischemic injury

Following permanent occlusion of the left middle cerebral artery (MCA) in rats, electrophysiological and hemodynamic characteristics of the periinfarct border zone were investigated in sham-operated (n = 6), untreated (n = 6), and MK-801-treated (3.0 mg/kg; n = 6) animals. For this purpose, direct current potential (DC), EEG, and blood flow (laser-Doppler flowmetry) were recorded from the cortex in the periphery of the MCA territory. In sham-operated rats, a single negative cortical DC deflection was observed after electrocoagulation of the cortex, whereas in untreated MCA-occluded animals, three to eight transient DC deflections were monitored during the initial 3 h of ischemia. The duration of these cortical DC shifts gradually increased from 1.2 ± 0.3 to 3.7 ± 2.7 min (mean ± SD; p < 0.05) during this time. In animals treated intraperitoneally with MK-801 (3.0 mg/kg) immediately after MCA occlusion, the number of cortical DC shifts significantly declined to one to three deflections (p < 0.005). The EEG of the treated animals revealed low-amplitude burst-suppression activity. In the untreated and treated experimental group, the reduction of cortical blood flow amounted to 69 ± 25 and 49 ± 13% of control, respectively. Despite the more pronounced cortical oligemia, MK-801 treatment resulted in a significant decrease of the volume of the ischemically injured tissue from 108 ± 38.5 (untreated group) to 58 ± 11.5 (p < 0.05) mm3. Our results suggest that repetitive cortical DC deflections in the periinfarct border zone contribute to the expansion of ischemic brain infarcts.

Cortical Negative DC Deflections following Middle Cerebral Artery Occlusion and KCl-Induced Spreading Depression: Effect on Blood Flow, Tissue Oxygenation, and Electroencephalogram

In the periphery of ischemic brain lesions, transient spreading depression-like direct current (DC) deflections occur that may be of pathophysiological importance for determining the volume of the ischemic infarct. The effect of these deflections on cerebral blood flow, tissue oxygen tension, and electrophysiology was studied in rats submitted to intraluminal thread occlusion of the middle cerebral artery (MCA) and compared with the changes following potassium chloride (KCl)-induced spreading depression of intact animals. Immediately after MCA occlusion, cortical laser–Doppler flow (LDF) in the periphery of the MCA territory sharply decreased to 35 ± 14% of control (mean ± SD; p < 0.05), tissue Po2 declined from 28 ± 4 to 21 ± 3 mm Hg (p < 0.05), and EEG power fell to ∼80% of control. During 7-h occlusion, 3–11 DC deflections with a mean duration of 5.2 ± 4.8 min occurred at irregular intervals, and EEG power gradually declined to 66 ± 16% of control (p < 0.05). During the passage of DC deflections, LDF did not change, but Po2 further declined to 19 ± 4 mm Hg (p < 0.05). KCl-induced depolarizations of intact rats were significantly shorter (1.4 ± 0.5 min; p < 0.05) and were accompanied by a 43% increase in LDF (p < 0.05) and a slight but significant increase in tissue Po2 from 22 ± 4 to 25 ± 4 mm Hg (p < 0.05). The comparison of periinfarct and KCl-induced depolarizations demonstrates that oxygen requirements are not coupled to an appropriate flow response in the periinfarct zone with severely reduced blood flow. The resulting episodes of relative hypoxia could explain the previously documented relationship between the number of depolarizations and infarct volume.

The effect of ischaemia and recirculation on protein synthesis in the rat brain.

Abstract— Rats were subjected to cerebral compression ischaemia for 15min and were subsequently recirculated with blood for periods up to 3 h. In vivo incorporation of intravenously administered L‐[1–14C]valine into total brain proteins was found to be severely inhibited (about 20% of controls) after 45 min of recirculation. After 3 h, protein synthesis had increased, the specific radioactivity of proteins then being about 40% of controls. The post‐ischaemic inhibition of protein synthesis was accompanied by a breakdown in polyribosomes and a concomitant increase in ribosomal subunits. In vitro incorporation of L‐[1–14C]phenylalanine by a postmitochondrial supernatant system derived from animals subjected to 15 min ischaemia and 15 min recirculation was also severely reduced and showed, in contrast to control animals, no response to the addition of a specific inhibitor of polypeptide chain initiation (Poly(I)). Together with the in vivo accumulation of ribosomal subunits this indicates a block in peptide chain initiation during the early stages of recirculation.

Reactive microglia in cerebral ischaemia: an early mediator of tissue damage?

Microglial cell activation is a rapidly occurring cellular response to cerebral ischaemia. Microglia proliferate, are recruited to the site of lesion, npregulate the expression of several surface molecules including major histocom‐patibility complex class I and II antigens, complement receptor and the amyloid precursor protein (APP) as well as newly expressed cytokines, e.g. interleukin‐1 and transforming growth factor pl. The ischaemia‐induced production of APP may contribute to amyloid deposition in the aged brain under conditions of hypofusion. Ultra‐structurally, microglia transform into phagocytes removing necrotic neurons but still respecting the integrity of eventually surviving neurons even in the close vicinity of necrotic neurons. Microglial activation starts within a few minutes after ischaemia and thus precedes the morphologically detectable neuronal damage. It additionally involves a transient generalized response within the first 24 hours post‐ischaemia even at sites without eventual neuronal cell death. In functional terms, the microglial reaction appears to be a double‐edged sword in ischaemia. Activated microglia may exert a cytotoxic effector function by releasing reactive oxygen species, nitric oxide, proteinases or inflammatory cytokines. All of these cytotoxic compounds may cause bystander damage following ischaemia. Pharmacological suppression of microglial activation after ischaemia has accordingly attenuated the extent of cell death and tissue damage. However, activated microglia support tissue repair by secreting factors such as transforming growth factor βl which may limit tissue damage as well as suppress astroglial scar formation. In line with ultrastructural observations microglial activation in ischaemia is a strictly controlled event. By secreting cytokines and growth factors activated microglia most likely serve seemingly opposed functions in ischaemia, i.e. maintenance as well as removal of injured neurons. Post‐ischaemic pharmacological modulation of microglial intervention in the cascade of events that lead to neuronal necrosis may help to improve the structural and functional outcome following CNS ischaemia.

Thrombolysis using plasminogen activator and heparin reduces cerebral no-reflow after resuscitation from cardiac arrest : an experimental study in the cat

ObjectiveSuccessful resuscitation of the brain requires complete microcirculatory reperfusion, which, however, may be impaired by activation of blood coagulation after cardiac arrest. The study addresses the question of whether postischemic thrombolysis is effective in reducing cerebral noreflow phenomenon.Design14 adult normothermic cats were submitted to 15-min cardiac arrest, followed by cardiopulmonary resuscitation (CPR) and 30 min of spontaneous recirculation. The CPR protocol included closed-chest cardiac massage, administration of epinephrine 0.2 mg/kg, bicarbonate 2mEq/kg per 30 min, and electrical defibrillation shocks.InterventionsDuring CPR, animals in the treatment group (n=6) received intravenous bolus injections of 100 U/kg heparin and 1 mg/kg recombinant tissue type plasminogen activator (rt-PA), followed by an infusion of rt-PA 1mg/kg per 30 min.Measurements and resultsMicrocirculatory reperfusion of the brain was visualized by labeling the circulating blood with 300 mg/kg of 15% fluorescein isothiocyanate albumin at the end of the recirculation period. Areas of cerebral noreflow — defined as the absence of microvascular filling — were identified by fluorescence microscopy at eight standard coronal levels of forebrain, and expressed as the percentage of total sectional area. One animal in the treatment group was excluded from further analysis because of intracerebral hemorrhage due to brain injury during trepanation. Autopsy revealed the absence of intracranial, intrathoracic, or intra-abdominal bleeding in all the other animals. In untreated animals (n=8), no-reflow affected 28±13% of total forebrain sectional areas, and only 1 out of 8 animals showed homogeneous reperfusion (i.e., no-reflow <15% of total forebrain sectional areas). Thrombolytic therapy (n=5) significantly reduced no-reflow to 7±5% of total forebrain sectional areas and all treated animals showed homogeneous reperfusion at the microcirculatory level.ConclusionsThe present data demonstrate that thrombolytic therapy improves microcirculatory reperfusion of the cat brain when administered during reperfusion after cardiac arrest.

Expression of transforming growth factor-β1 and interleukin-1β mRNA in rat brain following transient forebrain ischemia

Transforming growth factor-β1 (TGF-β1) and interleukin-1β mRNA expression were studied in rat brains after 30 min of global ischemia by in situ hybridization. Ischemia was produced by four-vessel occlusion followed by different recirculation times ranging between 15 min and 7 days. TGF-β1 mRNA could first be detected 3 days after ischemia in the hippocampus, in layers II/III of cortex, in the striatum and in parts of the ventral thalamus. At 7 days after recirculation a prominent increase in TGF-β1 mRNA was observed in the CA1 sector of the hippocampus. Induction of interleukin-1β mRNA, however, was less marked and limited to the rostral striatum 3 and 7 days after ischemia. TGF-β1 expression 7 days after ischemia correlated well with the histological localization of regions where neuronal degeneration and subsequent astrocytic and microglial activation had occurred. In adjacent brain sections, the distribution of TGF-β1 mRNA after 7 days closely resembled that of the immunostaining pattern of activated microglia, indicating that at this time point TGF-β1 mRNA was mainly produced by microglial cells. The late induction of TGF-β1 mRNA after ischemia points to an involvement in the persistent glial response rather than the initial glial activation. The differential pattern of interleukin-1β mRNA induction indicates regional variations of cytokine production after ischemic brain lesions.

[14C]leucine incorporation into brain proteins in gerbils after transient ischemia: relationship to selective vulnerability of hippocampus.

Abstract: Regional [14C]leucine incorporation into brain proteins was studied in gerbils after global ischemia for 5 min and recirculation times of 45 min to 7 days, using a combination of quantitative autoradiography and biochemical analysis. After recirculation for 45 min, incorporated radioactivity was reduced to ∼20–40% of control values in all ischemic brain regions. Specific activity of the tracer, in contrast, was increased, a finding indicating that the reduced incorporation of radioactivity was not due to reduced tracer influx from plasma or a dilution of the tracer by increased proteolysis. After recirculation for 6 h, [14C]leucine incorporation returned to control levels in all regions except the CA1 sector of the hippocampus, where it amounted to <50%. After 1 day, protein synthesis in the CA1 sector returned to ∼70% of control values, followed by a secondary decline to <50% after 3 days and returned to near control values after 7 days. Histological evaluations revealed selective neuronal death in the CA1 sector of the hippocampus after 3 days of recirculation. The complex time course of protein synthesis in the CA1 sector suggests a biphasic mode of injury, which may be related to similar changes of calcium homeostasis. The final return to near normal after CA1 neurons have disappeared is explained by astroglial proliferation and demonstrates that at this time protein synthesis is not a marker of neuronal viability.

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.

Effect of global system for mobile communication microwave exposure on the genomic response of the rat brain

The acute effect of global system for mobile communication (GSM) microwave exposure on the genomic response of the central nervous system was studied in rats by measuring changes in the messenger RNAs of hsp70, the transcription factor genes c-fos and c-jun and the glial structural gene GFAP using in situ hybridization histochemistry. Protein products of transcription factors, stress proteins and marker proteins of astroglial and microglial activation were assessed by immunocytochemistry. Cell proliferation was evaluated by bromodeoxyuridine incorporation. A special GSM radiofrequency test set, connected to a commercial cellular phone operating in the discontinuous transmission mode, was used to simulate GSM exposure. The study was conducted at time averaged and brain averaged specific absorption rates of 0.3 W/kg (GSM exposure), 1.5 W/kg (GSM exposure) and 7.5 W/kg (continuous wave exposure), respectively. Immediately after exposure, in situ hybridization revealed slight induction of hsp70 messenger RNA in the cerebellum and hippocampus after 7.5 W/kg exposure, but not at lower intensities. A slightly increased expression of c-fos messenger RNA was observed in the cerebellum, neocortex and piriform cortex of all groups subjected to immobilization, but no differences were found amongst different exposure conditions. C-jun and GFAP messenger RNAs did not increase in any of the experimental groups. 24 h after exposure, immunocytochemical analysis of FOS and JUN proteins (c-FOS, FOS B, c-JUN JUN B, JUN D), of HSP70 or of KROX-20 and -24 did not reveal any alterations. Seven days after exposure, neither increased cell proliferation nor altered expression of astroglial and microglial marker proteins were observed. In conclusion, acute high intensity microwave exposure of immobilized rats may induce some minor stress response but does not result in lasting adaptive or reactive changes of the brain.

Resuscitation potentials after prolonged global cerebral ischemia in cats.

Predictors of postischemic recovery were determined in adult cats subjected to one hour of global brain ischemia followed by blood recirculation for 3 h or longer. The incidence of postischemic recovery of evoked potentials and of spontaneous EEG activity was determined and related to the following variables: anesthesia (barbiturate or halothane/nitrous oxide), completeness of ischemia, body temperature, plasma glucose, arterial pH, blood gases, blood osmolality, postischemic BP, and cerebral blood flow (CBF). The closest correlation was obtained with the rate of initial postischemic recirculation; CBF above 40 ml/100 g. min resulted almost consistently in EEG recovery. Temperature, arterial pH, plasma glucose, and osmolality also influenced recovery. There was no correlation of EEG recovery with blood gases nor improvement of recovery after incomplete ischemia or barbiturate anesthesia. The results are interpreted in respect to common concepts of ischemic injury and the treatment of postischemic resuscitation disease.