Beat Alessandri

Lactate reduces glutamate-induced neurotoxicity in rat cortex

Experiments were carried out to test the hypothesis that lactate reduces the neurotoxicity of glutamate in vivo. MAP2 immunohistochemistry was used to measure lesion size, and microdialysis to measure the changes in glucose and lactate in the extracellular compartment. After implantation of a microdialysis probe 100 mM glutamate with or without 6 mM lactate was added to the perfusion medium and infused into the cortex of unanesthetized rats. Infusion of 100 mM glutamate for a period of 30 min produced a lesion of 6.05 ± 0.64 mm3, an increase in lactate of 124 ± 19% above basal and a 21 ± 9% reduction of glucose below basal level. When 6mM L‐lactate was perfused together with 100 mM glutamate there was a significant reduction in the size of the lesion and there was no reduction in dialysate glucose. When L‐lactate was replaced with D‐lactate the lesion size and the increase in dialysate lactate were greater than after glutamate alone. The neuroprotective role of L‐lactate is attributed to its ability to meet the increased energy demands of neurones exposed to high concentrations of glutamate.

Microdialytic monitoring during cerebrovascular surgery

Using microdialysis, levels of metabolites in the extracellular fluid of the cerebral cortex were monitored during neurovascular surgery (9 aneurysm and 5 extra-intracranial bypass operations). Our aim was to use microdialysis to detect any local ischemia which might be caused by brain retraction or temporary clipping. Parameters were therefore quantified whose levels in the dialysate are known to be influenced by ischemia (on-line pH, ascorbic acid, uric acid, glutathione, cysteine, glucose, lactate, glucose:lactate ratio). In the aneurysm series, on-line pH fell after introduction of the retractor, and in the majority of cases the other parameters also showed changes in accordance with ischemic conditions in the region of the probe. These changes disappeared at the end of retraction, or sometimes even before. During the bypass operations, there were no marked changes in on-line pH or in any of the measured parameters. However, in some of these patients values for the glucose:lactate ratio, ascorbic acid and uric acid lay outside the suggested basal levels for minimally disturbed cortex, indicating possible changes in metabolism caused by inadequate perfusion (carotid artery occlusion). We conclude that microdialysis is a sensitive method of detecting intraoperative changes in cerebral metabolism.

Extracellular levels of glucose and lactate measured by quantitative microdialysis in the human brain

Abstract The aim of this study was to use quantitative microdialysis to estimate the true extracellular concentrations of glucose and lactate in minimally disturbed human brain. These values are important as criteria for microdialytical monitoring in critical care patients and for determining therapy. Microdialysis procedures were carried out during tumor operations, the probe being inserted distant from the site of manipulation in minimally disturbed tissue. Two methods were used: 1. The zero net flux method of Lönnroth. 2. The low flow method (10 mm membrane length, flow rate 0.3 µl min-1, high in vivo recovery). Both methods gave similar values of about 2000 µM for lactate and slightly less for glucose (1700 µM). Glucose levels correspond with those measured by other methods in humans, allowing for the fact that our patients were anesthetised. Extracellular glucose levels were positively correlated with blood glucose values measured before the operation, and with extracellular lactate. Results confirm that extracellular glucose is zero when blood glucose is about 2 mM. [Neurol Res 2001; 23: 531-536]

Tempol, a novel stable nitroxide, reduces brain damage and free radical production, after acute subdural hematoma in the rat

Recent studies have shown that there is increased production of deleterious free radicals following acute subdural hematoma (ASDH). Scavenging them may therefore be of therapeutic benefit. Nitroxides are new, low molecular weight, cell permeable superoxide dismutase mimics. This study investigated the neuroprotective effect of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) following ASDH in the rat. Twenty-one male Sprague-Dawley rats were used in two studies: (1) a volumetric study of ischemic brain damage (n = 10); and (2) a microdialysis study measuring free radical generation after ASDH (n = 11). Ten minutes after induction of hematoma, the animals received 10 mg/kg Tempol or vehicle intravenously. In the volumetric study, 4 h after treatment, the rats were perfused, the brain removed, cut into serial 12-microm coronal sections, and stained. Ischemic areas were measured in eight predetermined stereotactic planes. In the microdialysis study, free radical production was measured using the salicylate trapping technique by quantifying 2,3-dihydrobenzoic acid (2,3-DHBA) and 2,5-DHBA using HPLC. In the volumetric study, Tempol treatment significantly reduced infarct volumes; 100.2 +/- 15.7 mm3 in Tempol-treated animals compared with 171.5 +/- 13.6 mm3 in controls (42% reduction, p = 0.0005). The microdialysis study demonstrated an early twofold increase of free radical production at 30 min, and returning to the baseline levels in controls. However, in Tempol-treated animals, this early surge was attenuated, and all measured values remained around the baseline levels throughout the experiments. Tempol thus provides significant neuroprotective effect in a rat model of ASDH, related to attenuation of superoxide radical production. The use of these low molecular weight, cell-permeable agents, which readily cross the blood-brain barrier and enter cells, thus appears indicated for acute pathologies, ASDH.

Effects of hypertonic/hyperoncotic treatment after rat cortical vein occlusion*

ObjectiveTo examine the effects of hypertonic/hyperoncotic treatment on physiologic variables and regional cerebral blood flow and to test its neuroprotective efficiency in a model of permanent venous ischemia. DesignRandomized prospective study. SettingUniversity research institute. SubjectsAdult male Wistar rats, weighing 359 ± 54 g (n = 38). InterventionsRats were subjected to photochemical occlusion of two adjacent cortical veins. A randomized infusion with vehicle (0.9% NaCl), 10% hydroxyethyl starch 200000 (HES), or 7.5% saline plus 10% hydroxyethyl starch 200000 (HHES) was started 30 mins after two-vein occlusion. Effects on physiologic variables and regional cerebral blood flow (assessed by laser Doppler flowmetry) were studied up to 120 mins after two-vein occlusion. Two days after occlusion, the brains were removed for histologic evaluation. Measurements and Main ResultsAfter occlusion, regional cerebral blood flow decreased by 50%, significantly in all groups (from 47.3 ± 3 to 22.2 ± 2.2 laser Doppler units). In the vehicle and HES groups, regional cerebral blood flow further decreased to 12.9 ± 1.9 and 17.8 ± 2.3 laser Doppler units, respectively. HHES improved regional cerebral blood flow significantly to 27.3 ± 3.5 laser Doppler units, particularly by reducing no-flow/low-flow areas and reducing infarct size. ConclusionWe found that HHES reduced infarct size as a consequence of an improved regional cerebral blood flow and reduced no-flow/low-flow areas in the tissue at risk in the two-vein occlusion model.

The Neuroprotective Effect of Lactate Is Not Due to Improved Glutamate Uptake after Controlled Cortical Impact in Rats

For many years lactate was considered to be a waste product of glycolysis. Data are accumulating that suggest that lactate is an important energy substrate for neurons during activation. In severe traumatic brain injury (TBI) glutamate release and ischemic cerebral blood flow (CBF) are major factors for a mismatch between energy demand and supply and for neuronal cell death. Although ATP and behavior could be improved by lactate treatment after TBI, no histological correlate nor any linkage to better astrocytic glutamate uptake or CBF as possible mechanisms have been described. We subjected male rats to a controlled cortical impact (CCI; 5 m/sec, 2.5 mm). To study the effects of lactate treatment on lesion volume, glutamate release, and CBF, animals were infused with either NaCl or 100 mM lactate for up to 3 h. The role of endogenous lactate was investigated by inhibiting transport with α-cyano-4-hydroxy-cinnamic acid (4-CIN; 90 mg/kg). Lactate treatment 15 min post-CCI reduced lesion volume from 21.1±2.8 mm³ to 12.1±1.9 mm³ at day 2 after CCI. Contusion produced a significant three- to fourfold increase of glutamate in microdialysates, but there was no significant difference between treatments that began 30 min before CCI. In this experiment lesion volume was significantly reduced by lactate at day 7 post-CCI (23.7±4 to 9.3±1-2 mm³). CBF increased immediately after CCI and dropped thereafter below baseline in all animals. Lactate infusion 15 min post-CCI elevated CBF for 20 min in 7 of 10 animals, whereas 7 of 8 NaCl-treated animals showed a further CBF decline. Neuroprotection was achieved by lactate treatment following contusion injury, whereas blocking of endogenous lactate transport exerted no adverse effects. Neuroprotection was not achieved by improved glutamate uptake into astrocytes, but was supported by augmented CBF following CCI. Due to its neuroprotective property, lactate might be a beneficial pharmacological treatment for TBI patients.

Acute and late changes in N-acetyl-aspartate following diffuse axonal injury in rats: an MRI spectroscopy and microdialysis study.

Abstract N-acetyl-aspartate (NAA) measured by proton nuclear magnetic resonance spectroscopy (1 H-NMR) has been used as a marker of neuronal injury in many cerebral pathologies. Therefore, we evaluate the roles of microdialysis vs. 1H-NMR as techniques to assess NAA (NAAd; NAA/Creatine ratio) in the living brain, and compare the results with whole brain NAA (NAAJ, analyzed by HPLC after diffuse traumatic brain injury (TBI). Acute (4 h post-injury survival) and late (48 h survival) changes were studied in a sham-operated group (Sham, n = 4), and two injured groups (TBI/4 h, n = 8; TBI/48 h, n = 7). Baseline NAAd was 8.17±1 iM, and there was no significant difference between groups. There was only a small (twice of control), but transient increase in NAAd in the TBI/4 h group after trauma. Baseline NAA/Cr ratio was 1.35± 0.2, which did not change significantly between baseline, 1, 2, 3, 4 and 48 h or between groups after TBI. Whole brain NAAW (baseline 8.5± 0.5 mmol kg~ wet weight) did not differ significantly between groups before and after TBI. Diffuse TBI did not produce long-term changes in NAA, assessed by three different methods. These results may indicate that NAA is not a sensitive marker of the severity of diffuse axonal damage. However, further studies are needed to evaluate whether confounding factors such as microdialysis probe, voxel position and non-regional tissue homogenization might have influenced our data. [Neurol Res 2000; 22: 705-712]

Acute Subdural Hematoma in Pigs: Role of Volume on Multiparametric Neuromonitoring and Histology

Traumatic brain injury (TBI) is often complicated by acute subdural hemorrhage (ASDH) with a high mortality rate. The pathophysiological mechanisms behind such an injury type and the contribution of blood to the extent of an injury remain poorly understood. Therefore, the goals of this study were to establish a porcine ASDH model in order to investigate pathomechanisms of ASDH and to compare effects induced by blood or sheer volume. Thus, we infused 2, 5, and 9 mL of blood (up to 15% of intracranial volume), and we compared a 5-mL blood and paraffin oil volume to separate out effects of extravasated blood on brain tissue. An extended neuromonitoring was applied that lasted up to 12 h after injury and included intracranial pressure (ICP), cerebral perfusion pressure (CPP), tissue oxygen concentration (ptiO(2)), biochemical markers (glutamate, lactate), somatosensory evoked potentials (SEP), brain water content, and histological assessment (Lesion Index [LI]). Volume-dependent changes were detected mainly during the first hours after injury. ICP increased to significant levels (p < 0.05) of 36.89 +/- 1.59, 15.52 +/- 0.48, and 11.25 +/- 0.35 mm Hg after 9, 5, and 2 mL of subdural blood, respectively (sham, 4.85 +/- 0.06 mm Hg). The ptiO(2) dropped drastically after 9 mL of subdural blood without recovery in both hemispheres to below 20% of baseline, but was affected little after 2 and 5 mL in the acute monitoring period (maximal drop to 71% of baseline). Later, 5 mL of blood led to a significant increase of ptiO(2) compared to 2 mL ipsilaterally (p < 0.05). Glutamate and lactate showed a comparable pattern with a long-lasting increase after 9 mL of blood and short-lasting changes after 2 and 5 mL. The two smaller volumes caused an increased brain swelling (2 mL, 80.60 +/- 0.34%; 5 mL, 81.20 +/- 0.66%; p < 0.05 vs. sham), a significant LI (sham, 6.4 +/- 1.4; 2 mL, 30.0 +/- 0.95; 5 mL, 32.1 +/- 1.2; p < 0.05 vs. sham), and a reduced SEP amplitude (5 mL, p < 0.05 vs. baseline) at the end of the experiment. A 9-mL led to herniation during the experiment causing dramatical brain swelling and acute histological damage. Comparison of blood volume with paraffin oil showed no significance, indicating that volume alone determines the acute pathophysiological processes leading to a rapidly developing histological damage. Additional effects due to blood contact with brain tissue (e.g., inflammation) may be detected only at later time points (>12 h).

Emboli Formation Rather Than Inflammatory Mediators Are Responsible for Increased Cerebral Water Content After Conventional and Assisted Beating-Heart Myocardial Revascularization in a Porcine Model

Background and Purpose— Emboli and proinflammatory mediators are suspected of generating cerebral edema after coronary surgery. In contrast to cardiopulmonary bypass (CPB), off-pump coronary artery bypass surgery (OPCAB) reduces microemboli count and proinflammatory mediator release but carries the risk of hemodynamic instability. A microaxial blood pump can augment cardiac output. Methods— Coronary bypasses were constructed in pigs with CPB and cardioplegia (n=9), OPCAB (n=9), or blood-pump support CAB (n=9). Nine animals underwent sham operation. Embolus count was monitored and regional cerebral blood flow was assessed with microspheres in 21 brain specimens per animal (n=189 per group). Interleukins 6 and 8 and tumor necrosis factor-&agr; concentrations were determined. These variables were studied before, during, and for 4 hours after surgery. Finally, cerebral water content was determined. Results— During CPB and blood-pump CAB, a significant number of emboli were counted in contrast to OPCAB and controls (P<0.05). During CPB, regional cerebral blood flow was affected (32 of 189) and showed reactive hyperemia except in 10 specimens after aortic cross-clamp release. This impairment persisted in 20 specimens. During and after OPCAB, regional cerebral blood flow remained nearly unchanged but showed low flow during (58 of 189) and after (35 of 189) the blood-pump run. A significant increase in proinflammatory mediators was observed only in the CPB group. CPB and blood-pump CAB significantly increased cerebral water content (P<0.05). A strong correlation between embolic load and cerebral water content was observed in all groups. No correlation between proinflammatory mediator release and cerebral water content was detected. Conclusions— Emboli formation rather than inflammatory mediators are responsible for increased cerebral water content after conventional and assisted beating-heart myocardial revascularization.

Effects of hypertonic/hyperoncotic treatment and surgical evacuation after acute subdural hematoma in rats

Objective: The treatment of acute subdural hematoma (ASDH) consists mainly of surgical evacuation of the hematoma. It is conceivable that early preoperative neuroprotection with hypertonic/hyperoncotic treatment (HHT) can improve survival rates. The present study investigated the benefit of treatment with hypertonic/hyperoncotic solution on functional and histologic outcome as supportive therapy accompanying surgical intervention. Design: Laboratory experiment. Setting: University laboratory. Subjects: Male Sprague-Dawley rats weighing 296–350 g (n = 56). Interventions: ASDH was induced through subdural infusion of 400 &mgr;L of autologous venous blood. Thirty minutes after subdural blood infusion, the rats received either HyperHAES (7.2% saline/6% hydroxyethyl starch) or vehicle (NaCl 0.9%) intravenously, followed by surgical evacuation of the hematoma 1 hr after ASDH induction in those rats scheduled for surgical treatment. The experiment was divided into two parts: an acute study, which explored acute effects of HHT on blood variables, ASDH-induced changes of intracranial pressure (ICP), and cerebral perfusion pressure (CPP), and a chronic study, which investigated the chronic effects of HHT, surgical blood clot evacuation, and the combination of both on the functional and histologic outcome following ASDH (12 days). Measurements and Main Results: In the acute study, HHT expectedly raised the serum sodium concentration and lowered hematocrit. ASDH increased ICP and decreased CPP in all groups. HHT improved CPP by reducing ICP. In the chronic study, all treated groups showed a better recovery with respect to neurologic function and neuronal cell death compared with the vehicle-treated ASDH group. HHT with surgical evacuation or HHT alone improved functional and histologic outcome slightly more than surgical evacuation alone. Conclusions: In this rat model, HHT led to a decrease of ICP after ASDH. This significantly improved functional and histologic outcome, which was comparable to the effects after blood evacuation alone. The combination of evacuation of subdural blood and early HHT improved histologic outcome further but not significantly, which was due to the strong effects of single treatments and a ceiling effect of the combined treatment in this model.