Axel Heimann

In vitro and in vivo porphyrin accumulation by C6 glioma cells after exposure to 5-aminolevulinic acid

Several malignant tissues synthesize endogenous porphyrins after exposure to 5-aminolevulinic acid (5-ALA). The present experiments have been designed to elucidate whether the C6 glioma cell, a model cell for human malignant glioma, similarly synthesizes porphyrins when exposed to 5-ALA, and whether specific synthesis occurs when C6 cells are inoculated into rat brains to form a tumor. In this situation the blood-brain barrier may interfere with 5-ALA availability, and spreading of porphyrins with edema outside the tumor may occur. Flow cytometry is used to determine the course of cell volume and porphyrin fluorescence intensities in cultured C6 cells which are incubated in 1 mM 5-ALA. For the induction of experimental brain tumors, 10(4) untreated C6 cells are inoculated into the brains of rats. After 9 days animals receive 100 mg 5-ALA/kg body weight. Brains are removed after 3, 6, or 9 h and frozen coronal sections obtained for H/E staining or fluorescence spectography. Cultured C6 cells show a linear increase of protoporphyrin IX fluorescence after exposure to 5-ALA, which begins to plateau after 85 min. Marked fluorescence is also observed in solid and infiltrating experimental tumor. However, faint fluorescence also occurs in normal tissue, basal pia, choroid plexus, and, more obviously, in white-matter tracts bordering the tumor (maximal distance: 1.5 +/- 0.7 mm). The observations demonstrate that C6 cells synthesize protoporphyrin IX after exposure to 5-ALA in vitro and in vivo. However, when utilizing 5-ALA for fluorescence detection or photodynamic therapy of brain tumors, attention should be paid to the possibility of protoporphyrin IX occurring outside the tumor.

Intracoronary Application of C1 Esterase Inhibitor Improves Cardiac Function and Reduces Myocardial Necrosis in an Experimental Model of Ischemia and Reperfusion

BACKGROUND Myocardial injury from ischemia can be aggravated by reperfusion of the jeopardized area. The precise underlying mechanisms have not been clearly defined, but proinflammatory events, including complement activation, leukocyte adhesion, and infiltration and release of diverse mediators, probably play important roles. The present study addresses the possibility of reducing reperfusion damage by the application of C1 esterase inhibitor (C1-INH). METHODS AND RESULTS Cardioprotection by C1-INH 20 IU/kg IC was examined in a pig model with 60 minutes of coronary occlusion, followed by 120 minutes of reperfusion. C1-INH was administered during the first 5 minutes of coronary reperfusion Compared with the NaCl controls, C1-INH reduced myocardial injury (48.8 +/- 7.8% versus 73.4 +/- 4.0% necrosis of area at risk, P < or = .018). C1-INH treatment significantly reduced circulating C3a and slightly attenuated C5a plasma concentrations. Myocardial protection was accompanied by reduced plasma concentration of creatine kinase and troponin-T. C1-INH had no effect on global hemodynamic parameters, but local myocardial contractility was markedly improved in the ischemic zone. In the short-axis view, 137 degrees of the anteroseptal region showed significantly improved wall motion at early and 29 degrees at late reperfusion with C1-INH treatment. CONCLUSIONS C1-INH significantly protects ischemic tissue from reperfusion damage, reduces myocardial necrosis, and improves local cardiac function.

Neuroprotection of S(+) ketamine isomer in global forebrain ischemia

The non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist ketamine can block the action of excitotoxic amino acids in the central nervous system. S(+) ketamine has a 2-3 times higher anesthetic potency compared with the ketamine-racemate and also shows a higher neuroprotective efficacy in vitro. To determine the neuroprotective activity of S(+) ketamine compared with its R(-) stereoisomer in vivo, we examined the functional and neurohistological outcome in rats treated 15 min after global forebrain ischemia with S(+) ketamine in different dosages compared with R(-) ketamine. Influence of the treatment on regional cerebral blood flow (rCBF) and cortical oxygen saturation (HbO2) was monitored over 1 h after the ischemia using laser doppler flowmetry and microphotospectrometry respectively. Sixty and ninety mg/kg of S(+) ketamine but not R(-) ketamine significantly reduced neuronal cell loss in the cortex compared with the saline treated group. No significant neuroprotection was observed in the hippocampus. Although no significant change in rCBF was found, S(+) ketamine restored the cortical HbO2 to preischemic values. These results indicate that S(+) ketamine in higher dosages can reduce neuronal damage in the cortex after cerebral ischemia, possibly by improving the ratio of oxygen supply to consumption in the postischemic tissue.

Cerebral blood flow alterations in a rat model of cerebral sinus thrombosis.

Background and Purpose: Outcome from sinus vein thrombosis is very variable, with symptoms from headache to coma. Experimental findings suggest that an involvement of cortical veins is necessary to affect the cerebral microcirculation. Laser Doppler flowmetry was used to investigate the regional and temporal changes in local cortical blood flow after experimental occlusion and thrombosis of the superior sagittal sinus and tributary cortical veins in rats. Methods: Thrombosis was induced by slow injection of kaolin‐cephalin suspension after frontal and caudal ligation of the sagittal sinus in rats. Local cerebral blood flow was measured by laser Doppler flowmetry and correlated with parenchymal damage found 24 hours after induction of thrombosis. Results: Local cerebral blood flow 1 hour after sinus occlusion and induction of thrombosis had decreased to 60.92±29.05% (p <0.01); however, there was a large variability among individual animals. Only five of 12 rats showed histological damage and intracerebral hemorrhages 24 hours after induction of thrombosis. A subgroup analysis revealed that parenchymal damage occurred in concurrence with reduced blood flow values after sinus ligation and injection of the thrombogenic material. Sinus thrombosis alone, without alteration of blood flow, did not cause tissue necrosis. Conclusions: The data support the contention that sinus vein thrombosis evolves gradually, with major symptoms occurring only if the thrombus expands from the sinus into bridging and cortical veins. Collateral venous outflow pathways are thereby occluded, and local blood flow may become reduced to and below the ischemic threshold. (Stroke 1993;24:563‐570)

Cerebral Blood Flow Autoregulation During Hypobaric Hypotension Assessed by Laser Doppler Scanning

Hypobaric hypotension was used to reduce systemic blood pressure in rats below the lower threshold of CBF autoregulation to evaluate a new laser Doppler (LD) “scanning” technique. Spontaneously breathing male Wistar Kyoto rats (n = 8) were anesthetized with chloral hydrate and the head fixed in a stereotaxic head holder. A cranial window with intact dura mater was introduced to assess local CBF (lCBF) by LD. One stationary probe served to detect rapid flow changes, whereas the second probe was used to sample lCBF recordings from many cortical locations by means of a stepping motor-controlled micromanipulator to obtain lCBF frequency histograms. Advantages are an improved spatial resolution together with the easy detection of low-flow areas and a better comparison of data from individual experiments. Arterial blood pressure was stepwise reduced by exposing the lower body portions to subatmospheric pressures (hypobaric hypotension), thus avoiding the use of drugs or heparinization. The lower threshold of CBF autoregulation was detected by “scanning” at arterial pressures between 50 and 46 mm Hg, with low-flow spots occurring immediately. The data suggest LD scanning as a method suited particularly for studies where lCBF inhomogeneities are expected, e.g., the ischemic penumbra or sinus vein thrombosis.

Early albumin infusion improves global and local hemodynamics and reduces inflammatory response in hemorrhagic shock.

Objective To evaluate the effects of an early, short-term albumin infusion on mesenteric microcirculation and global hemodynamics in hemorrhagic shock. Design A prospective, randomized study. Setting Animal laboratory at a university medical clinic. Subjects Seventeen Sprague-Dawley rats weighing 250–400 g. Interventions The rats underwent median laparotomy and exteriorization of an ileal loop for intravital microscopy of the mesenteric microcirculation. Volume-controlled hemorrhagic shock was provoked by arterial blood withdrawal (2.5 mL/100 g body weight for 60 mins), followed by a 4-hr reperfusion period. Albumin (20%) or 0.9% NaCl was administered intravenously as a continuous infusion for 30 mins at the beginning of reperfusion. Reperfusion time mimicked a “prehospital” phase of 30 mins followed by a quasi “in-hospital” phase of 3.5 hrs. The “in-hospital” phase in both groups was initiated by substitution of blood followed by reperfusion with normal saline. Measurements and Main Results Central hemodynamics, mesenteric microcirculation, and arterial blood gas parameters were monitored before, during, and 60 mins after hemorrhagic shock, and for a 240-min follow-up period after initiation of reperfusion. Application of albumin markedly reduced rolling and adherent leukocytes, maximum velocity, and shear rate in the mesenteric microcirculation. Later, after improvement of mesenteric microcirculation, an intermittent increase of central venous pressure and abdominal blood flow and decrease of hematocrit was observed. Conclusions Albumin treatment of hemorrhagic shock improves microcirculation and global hemodynamics and attenuates the inflammatory response to reperfusion. It may provide clinical benefit when applied at an early stage of reperfusion during hemorrhagic shock.

Local Cerebral Blood Flow in a Rat Cortical Vein Occlusion Model

The symptoms following sinus and vein occlusion observed in patients and experimental animals display a considerable variability that so far remains largely unexplained. In a rat cortical vein occlusion model using a photochemical thrombotic technique, we examined changes in the cerebral venous flow pattern by fluorescence angiography and regional cerebral blood flow (rCBF) and cerebral blood volume fraction (CBVF) by a modern laser Doppler “scanning” technique. Brain damage was assessed histologically. Fluorescence angiographic findings fell into two groups: group A, rats with an altered venous flow pattern after occlusion (n = 12), and group B, rats with interruption of blood flow and/or a growing venous thrombus (n = 5). In addition, sham-operated animals made up group C (n = 5). Extravasation of fluorescein, a massive decrease in rCBF, a short-lasting increase in CBVF, and regional brain damage were typical for group B. In addition, cortical CBF mapping revealed a transient hyperperfusion zone with hyperemia surrounding a hypoperfused ischemic core in group B. A circulation perturbation following venous occlusion appeared near those occluded cerebral veins without sufficient collateral flow. Furthermore, the venous thrombus continued to grow, accompanied by local critical ischemia and severe brain damage. Conversely, 71% of the animals (12 of 17) tolerated occlusion of a solitary vein without major flow disturbances or histological evidence of damage to the CNS (group A).

Capillary Flow and Diameter Changes during Reperfusion after Global Cerebral Ischemia Studied by Intravital Video Microscopy

The reaction of cerebral capillaries to ischemia is unclear. Based on Hossmanns observation of postischemic “delayed hypoperfusion,” we hypothesized that capillary flow is decreased during reperfusion because of increased precapillary flow resistance. To test this hypothesis, we measured cerebral capillary erythrocyte velocity and diameter changes by intravital microscopy in gerbils. A cranial window was prepared over the frontoparietal cortex in 26 gerbils anesthetized with halothane. The animals underwent either a sham operation or fifteen minutes of bilateral carotid artery occlusion causing global cerebral ischemia. Capillary flow velocities were measured by frame-to-frame tracking of fluorescein isothiocyanate labeled erythrocytes in 1800 capillaries after 1-hour reperfusion. Capillary flow velocities were decreased compared to control (0.25 ± 0.27mm/s vs. 0.76 ± 0.45 mm/s; P < 0.001). Precapillary arteriole diameters in reperfused animals were reduced to 76.3 ± 6.9% compared to baseline (P < 0.05). Capillary diameters in reperfused animals (2.87 ± 0.97 μm) were reduced (P < 0.001) compared to control (4.08 ± 1.19 μm). Similar reductions of precapillary (24%) and capillary vessel diameters (30%) and absolute capillary flow heterogeneity indicate that delayed (capillary) hypoperfusion occurs as a consequence of increased precapillary arteriole tone during reperfusion.

Laser-Doppler Scanning of Local Cerebral Blood Flow and Reserve Capacity and Testing of Motor and Memory Functions in a Chronic 2-Vessel Occlusion Model in Rats

BACKGROUND AND PURPOSE An animal model of incomplete forebrain ischemia resembling human hemodynamic insufficiency was established. The model allows examination of acute and chronic changes of local cerebral blood flow (lCBF) and reserve capacity in correlation with behavioral parameters. METHODS Anesthetized male Wistar-Kyoto rats underwent bilateral carotid occlusion (BCO). Laser-Doppler scanning of lCBF at baseline conditions and after acetazolamide was done 30 minutes after BCO, motor and memory function tests were administered after 1 and 2 days, and both investigations were repeated after 1, 2, 4, and 6 weeks. A sham-operated and a control group without any vessel manipulation served as controls. RESULTS lCBF dropped within 60 minutes after surgery by 62% (P<0.001) in 10 animals surviving BCO (BCOsurvival) and by 69% in 5 rats that died within 9 days (BCOlethal). Acetazolamide increased lCBF to 142.33% in controls, to 136.66% in sham-operated rats (both significant), and to 104.80% in BCOsurvival (not significant), and it decreased flow by 23.1% in BCOlethal rats (P<0.001). Baseline lCBF normalized within 4 weeks. Total motor function scores were significantly reduced from 9 points preoperatively to 5.80+/-0.65 in BCOlethal and 6.68+/-0.54 points in BCOsurvival rats 1 day after occlusion. Memory retention function remained impaired after BCO, as did the acetazolamide response, which correlated with motor score and was inversely related to maze exploration time. CONCLUSIONS This model allows long-term follow-up of cerebral function, lCBF, and reserve capacity in a pathophysiological setting similar to hemodynamic insufficiency in humans.

Brain oxygen monitoring: in-vitro accuracy, long-term drift and response-time of Licox- and Neurotrend sensors

SummaryBackground. Oxygen tension sensors have been used to monitor tissue oxygenation in human brain for several years. The working principals of the most frequently used sensors, the Licox (LX) and Neurotrend (NT), are different, and they have never been validated independently for correct measurement in vitro. Therefore, we tried to clarify if the two currently available sensors provide sufficient accuracy and stability.Method. 12 LX oxygen tension sensors and NT sensors were placed into a liquid-filled tonometer chamber. The solution was kept at 37 ± 0.2 °C and equilibrated with five calibration gases containing different O2- and CO2-concentrations. After equilibration, readings were taken for each gas concentration (accuracy test). Afterwards, the sensors were left in 3% O2 and 9% CO2 and readings were taken after 24, 48, 72, 96 and 120 hours (drift test). Thereafter, a 90% response time test was performed transferring sensors from 1% to 5% oxygen concentration and back, using pre-equilibrated tonometers.Findings. All Licox oxygen probes [12] were used for this study. Two of 14 Neurotrend sensors did not calibrate, revealing a failure rate of 14% for NT. Oxygen tension during the accuracy test was measured as follows: 1% O2 (7.1 mmHg): LX 6.5 ± 0.4, NT 5.3 ± 2.3 mmHg, 2% O2 (14.2 mmHg): LX 12.9 ± 0.6, NT 12.1 ± 2.2 mmHg, 3% O2 (21.4 mmHg): LX 19.8 ± 0.7, NT 19.4 ± 2.4 mmHg, 5% O2 (35.8 mmHg): LX 33.4 ± 1.0 mmHg, NT 33.5 ± 2.9 mmHg, 8% O2 (57.0 mmHg): 53.8 ± 1.5, NT 53.6 ± 3.3 mmHg. After 120 hours in 3% O2 (21 mmHg), LX measured 19.8 ± 1.9 mmHg, NT 17.9 ± 4.7 mmHg. 90% response time from 1% to 5%/5% to 1% oxygen concentration was 129 ± 27/174 ± 26  sec for LX, 55 ± 19/98 ± 39 sec for NT.Conclusions. Both systems are measuring oxygen tension sufficiently, but more accurately with LX probes. NT sensors read significantly lower pO2 in 1% O2 and show an increasing deviation with higher oxygen concentrations which was due to two of twelve probes. A slight drift towards lower oxygen tension readings for both sensors but more pronounced for the NT does not impair long-term use. NT measures pCO2 and pH very accurately.