Oliver Kempski

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.

Swelling, Acidosis, and Irreversible Damage of Glial Cells from Exposure to Arachidonic Acid in vitro

Swelling and damage of C6 glioma cells and of primary cultured astrocytes were analyzed in vitro during incubation with arachidonic acid (AA; 20:4). The cells were suspended in a physiological medium supplemented with AA at concentrations of 0.001–1.0 mM. Cell swelling was quantified by flow cytometry with hydrodynamic focusing. Flow cytometry was also utilized for assessment of cell viability by exclusion of the fluorescent dye propidium iodide and for measurement of the intracellular pH (pHi) by 2′,7′-bis-(2-carboxyethyl)−5(and −6)carboxyfluorescein. Administration of AA caused an immediate dose-dependent swelling of C6 glioma cells, even at a concentration of 0.01 mM. At this level cell volume increased within 20 min to 105.0% of control, at 0.1 mM to 111.0%, while at 1.0 mM to 123.7%. Following a phase of rapid cell volume increase, swelling leveled off during the subsequent observation period of 70 min. Viability of the C6 glioma cells was 90% under control conditions. It remained unchanged after raising AA concentrations to 0.1 mM. At 0.5 mM, however, cell viability fell to 72.8%, and at 1.0 mM to 32.7%. pHi of the glioma cells was 7.3 under control conditions. In parallel with the early swelling phase, AA led to a dose-dependent decrease of the intracellular pH and an elevated lactate production of the cells. During incubation with 0.1 mM AA, pHi decreased to 7.06 after 5 min, but recovered to normal subsequently. In addition, swelling-inducing properties of linoleic (18:2) or stearic (18:0) acid were analyzed for evaluation of the specificity of glial swelling induced by AA. Whereas stearic acid (0.1 mM) failed to induce a swelling response, linoleic acid (0.1 mM) was found to be effective. The volume increase of the glial cells, however, was only half of that found during exposure to AA at the same concentration. Further, glial swelling from AA or linoleic acid was completely inhibited by the aminosteroid U-74389F, an antagonist of lipid peroxidation. Finally, omission of Na+ ions in the suspension medium with replacement by choline led also to inhibition of the cell volume increase by AA. Experiments using astrocytes from primary culture confirmed the swelling-inducing properties of AA at a quantitative level, whereas vulnerability of the cells to AA was increased. The present results demonstrate an important role of AA in cytotoxic swelling and irreversible damage of glial cells at concentrations that occur in vivo in cerebral ischemia or trauma. The damaging potential of AA might be enhanced by a concurrently evolving intracellular acidosis, stimulating the formation of oxygen-derived free radicals and lipid peroxidation.

Mediators of brain edema and secondary brain damage.

Progress in our understanding of the roles of vasogenic and cytotoxic brain edema in secondary brain damage can be expected from studies of the ability of biochemical factors to open the blood-brain barrier, derange the microcirculation, and cause cell swelling and necrosis. Mediator compounds are considered to form or to become released in an area of primarily damaged brain (necrosis) and to enter the cerebral parenchyma through the broken blood-brain barrier from the intravascular space. Many biochemical factors must be considered. We suggested three criteria for determining the roles of mediators: a) they must inflict brain tissue damage, b) they must occur in pathologic concentrations or in compartments not normally present, and c) specific inhibition should attenuate secondary brain damage. These requirements are met by the kallikrein-kinin system and by glutamate. In the case of arachidonic acid and its many metabolites, the concept is difficult to test because fatty acids may be active only if not bound to proteins, and therapeutic inhibition might be difficult. A variety of mediators may enhance each other in a cascade manner by various initiating reactions that might be amenable for pharmacologic inhibition.

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 sinus and venous thrombosis in rats induces long-term deficits in brain function and morphology. Evidence for a cytotoxic genesis

The pathophysiology of cerebral venous infarctions is poorly understood, due partially to the lack of a suitable experimental model. Therefore, we developed a model in rats to study acute and long-term changes of brain function and morphology following thrombosis of the superior sagittal sinus. The superior sagittal sinus of rats was exposed, ligated, and injected with thrombogenic material. Thrombosis of the longitudinal sinus and ascending cortical veins was monitored by intravital fluorescence angiography. Histology was studied at 24 h and 4 weeks after thrombosis and changes in intracranial pressure, electroencephalogram (EEG), and tissue impedance were noted. Spontaneous locomotor activity was followed for 4 weeks after thrombosis. The effect of heparin treatment on tissue impedance was evaluated. Thrombosis of the superior sagittal sinus could be regularly induced, although pathological sequelae developed only if ascending veins were affected. Sinus and venous thrombosis was histologically characterized by bilateral, parasagittal infarctions. Thrombosis induction was followed by an increase in intracranial pressure from 4.7 ± 1.6 to 12.8 ± 2.4 mm Hg (n = 4) at 1 h after thrombosis, associated with an exponential rise in tissue impedance to 165 ± 14% (n = 8) of the control. EEG changes were similar to those following global cerebral ischemia and remained pathological for up to 6 months after thrombosis (n = 6). As a permanent behavioral deficit spontaneous locomotor activity was reduced to 60 ± 10% (n = 6) of the control. Finally, the administration of heparin (1 IU/g body weight) after thrombosis induction was found to reverse the pathological tissue impedance response of the brain. In conclusion, involvement of ascending cortical veins following sinus thrombosis appears to be critical for the development of irreversible tissue damage, such as infarction. Changes in intracranial pressure and tissue impedance suggest that the venous thrombosis was followed by brain edema of a predominantly cytotoxic nature. Venous thrombosis led to long-term changes of brain function, as demonstrated by persistent disturbances of the EEG or of the spontaneous locomotor drive. These deficits may be amenable to treatment with heparin.

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)

Glial Ion Transport and Volume Control

K(+)-induced glial swelling results from an intricate interaction of transport and diffusion processes and metabolic stimulation, with many open questions remaining. Our concept of the major mechanisms involved can be summarized as follows: high extracellular K+ causes a burst-like stimulation of Na+/K+ ATPase and, hence, increases the metabolic demands. Lactate is produced; the cell is slightly acidified. To maintain a normal intracellular pH, the Na+/K+ antiporter extrudes protons and supplies Na+ for further Na+/K+ exchange. In addition, K+ ions enter the cell via membrane channels or furosemide-inhibitable transport. K+, Cl-, and lactate- ions accumulate as the osmotic basis for cell swelling. Later, cell volume normalizes slowly, a process involving lactate export and other, so far unidentified mechanisms. Taken together, the temporary swelling of glia at high K+ concentrations is the result of a homeostatic function, for the maintenance of a constant extracellular potassium concentration. Ion control ranges over volume control. In pathophysiologic states the loss of cell volume regulation may become a clinical problem, if cerebral swelling leads to an increase in intracranial pressure. It should be kept in mind, however, that elevation of the extracellular K+ concentration is not the only cause of glial swelling. Tissue acidosis, the release of neurotransmitters, especially glutamate, or free fatty acids are other mediator mechanisms initiating the swelling of glial elements. Only under controlled in vitro conditions can the individual significance of these factors be evaluated on a quantitative basis. Therapeutic approaches should be selected very carefully in order to maintain homeostatic mechanisms that are of utmost importance, especially after an insult to the brain.

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.