John F. Stover

Early expression of glutamate transporter proteins in ramified microglia after controlled cortical impact injury in the rat

Traumatic brain injury is followed by increased extracellular glutamate concentration. Uptake of glutamate is mainly mediated by the glial glutamate transporters GLAST and GLT‐1. Extent and distribution of GLAST and GLT‐1 were studied in a rat model of controlled cortical impact injury (CCII). Western Blot analysis revealed lowest levels of GLAST and GLT‐1 with a decrease by 40%–54% and 42%–49% between 24 and 72 h posttrauma. By 8 h after CCII, CSF glutamate levels were increased (10.5 μM vs. 2.56 μM in controls; P < 0.001), reaching maximum values by 48 h. A significant increase in de novo GLAST and GLT‐1 expressing ramified microglia was observed within 4 h, reached a stable level by 48 h, and remained high up to 72 h after CCII. Furthermore, ramified microglia de novo expressed the neuronal glutamate transporter EAAC1 after CCII. Following CCII, GLAST/GLT‐1 and GFAP coexpressing astrocytes were immediately reduced, reaching minimum levels within 8 h. This reduction of expression could be either due to protein downregulation or loss of astrocytes. At 72 h, a marked population of GLAST‐ and GLT‐1–positive reactive astrocytes appeared. These results support the hypothesis that reduced astrocytic GLAST and GLT‐1 protein levels following CCII contribute to evolving secondary injury. Microglia are capable of de novo expressing glutamate transporter proteins, indicating that the expression of glial and neuronal glutamate transporters is not restricted to a specific glial or neuronal lineage. Ramified microglia may play an important compensatory role in the early regulation of extracellular glutamate after CCII. GLIA 35:167–179, 2001.

Effects of early and late intravenous norepinephrine infusion on cerebral perfusion, microcirculation, brain-tissue oxygenation, and edema formation in brain-injured rats.

ObjectivesReduction of cerebral perfusion during the early phase after traumatic brain injury is followed by a later phase of normal to increased perfusion. Thus, pharmacologically elevating mean arterial blood pressure with the aim of improving cerebral perfusion may exert different time-dependent effects on cortical perfusion, microcirculation, tissue oxygenation and brain edema formation after traumatic brain injury. DesignRandomized, placebo-controlled trial. SettingExperimental laboratory at a university hospital. SubjectsA total of 37 male Sprague-Dawley rats subjected to a focal cortical contusion. InterventionsAt 4 or 24 hrs after focal traumatic brain injury, mean arterial blood pressure was increased to 120 mm Hg for 90 mins by infusing norepinephrine. In rats receiving physiologic saline, mean arterial blood pressure remained unchanged. In the first series, pericontusional cortical perfusion was measured using the laser Doppler flowmetry scanning technique before injury and before, during, and after the infusion period. In a second series, intracranial and cerebral perfusion pressure and intraparenchymal perfusion and tissue oxygen measured within the contused and pericontusional cortex were recorded continuously before, during, and after norepinephrine infusion. Changes in cortical microcirculation were investigated by orthogonal polarization spectral imaging. At the end of each experiment, hemispheric swelling and water content were determined gravimetrically. Measurements and Main ResultsAt 4 and 24 hrs after traumatic brain injury, intravenous norepinephrine significantly increased pericontusional cortical perfusion, which was also reflected by an increase in diameters and flow velocities of pericontusional arterioles and venules. Cerebral perfusion pressure and intraparenchymal perfusion and tissue oxygen were significantly increased during norepinephrine infusion at 4 and 24 hrs. Hemispheric swelling and water content showed no difference between the groups. ConclusionsAfter cortical impact injury, early and late intravenous norepinephrine infusion pressure-dependently increased cerebral perfusion and tissue oxygenation without aggravating or reducing brain edema formation. Future studies are warranted to determine long-term changes of short and prolonged norepinephrine-induced increases in mean arterial blood pressure and cerebral perfusion pressure.

Relation of cerebral energy metabolism and extracellular nitrite and nitrate concentrations in patients after aneurysmal subarachnoid hemorrhage

In a prospective clinical investigation on neurochemical intensive care monitoring, the authors aim was to elucidate the temporal profile of nitric oxide metabolite concentrations—that is, nitrite and nitrate (NOx)—and compounds related to energy-metabolism in the cerebral interstitium of patients after aneurysmal subarachnoid hemorrhage (SAH). During aneurysm surgery, microdialysis probes were implanted in cerebral white matter of the vascular territory most likely affected by vasospasm. Temporal profiles of NOx were analyzed in a subset of 10 patients (7 female, 3 male, mean age = 47 ± 14 years). Microdialysis was performed for 152 ± 63 hours. Extracellular metabolites (glucose, lactate, pyruvate, glutamate) were recovered from the extracellular fluid of the cerebral parenchyma. NOx was measured using a fluorometric assay. After early surgery, SAH patients revealed characteristic decreases of NOx from initial values of 46.2 ± 34.8 μmol/L to 23.5 ± 9.0 μmol/L on day 7 after SAH (P < 0.05). Decreases in NOx were seen regardless of development of delayed ischemia (DIND). Overall NOx correlated intraindividually with glucose, lactate, and glutamate (r = 0.58, P < 0.05;r = 0.32, P < 0.05;r = 0.28, P < 0.05; respectively). After SAH, cerebral extracellular concentrations of NO metabolites decrease over time and are associated with concomitant alterations in energy-or damage-related compounds. This could be related to reduced NO availability, potentially leading to an imbalance of vasodilatory and vasoconstrictive factors. On the basis of the current findings, however, subsequent development of DIND cannot be explained by a lack of vasodilatory NO alone.

Influence of Norepinephrine and Dopamine on Cortical Perfusion, EEG Activity, Extracellular Glutamate, and Brain Edema in Rats after Controlled Cortical Impact Injury

Following traumatic brain injury, catecholamines given to ameliorate cerebral perfusion may induce brain damage via cerebral arteriolar constriction and increased neuronal excitation. In the present study the acute effects of norepinephrine and dopamine on pericontusional cortical perfusion (rCBF), electroencephalographic (EEG) activity, extracellular glutamate, and brain edema were investigated in rats following controlled cortical impact injury (CCI). rCBF, cerebral perfusion pressure (CPP), EEG activity, and glutamate were determined before, during, and after infusing norepinephrine or dopamine, increasing MABP to 120 mm Hg for 90 min at 4 h after CCI. Control rats received physiological saline. At 8 h after CCI, hemispheric swelling and water content were determined gravimetrically. Following CCI, rCBF was significantly decreased. In parallel to elevating MABP and CPP, rCBF was significantly increased by norepinephrine and dopamine, being mostly pronounced with norepinephrine (+44% vs. +29%). In controls, rCBF remained diminished (-45%). EEG activity was significantly increased by norepinephrine and dopamine, while pericontusional glutamate was only elevated by norepinephrine (28 +/- 6 vs. 8 +/- 4 microM). Brain edema was not increased compared to control rats. Despite significantly increasing MABP and CPP to the same extent, norepinephrine and dopamine seem to differentially influence pericontusional cortical perfusion and glutamatergic transmission. In addition to the pressure-passive increase in CPP local cerebral effects seem to account for the sustained norepinephrine-induced increase in pericontusional cortical perfusion. The significantly elevated pericontusional glutamate concentrations in conjunction with the increased EEG activity suggest a sustained metabolically driven increase in cortical perfusion during norepinephrine infusion.

In vitro norepinephrine significantly activates isolated platelets from healthy volunteers and critically ill patients following severe traumatic brain injury

IntroductionNorepinephrine, regularly used to increase systemic arterial blood pressure and thus improve cerebral perfusion following severe traumatic brain injury (TBI), may activate platelets. This, in turn, could promote microthrombosis formation and induce additional brain damage.MethodsThe objective of this study was to investigate the influence of norepinephrine on platelets isolated from healthy volunteers and TBI patients during the first two post-traumatic weeks. A total of 18 female and 18 male healthy volunteers of different age groups were recruited, while 11 critically ill TBI patients admitted consecutively to our intensive care unit were studied. Arterial and jugular venous platelets were isolated from norepinephrine-receiving TBI patients; peripheral venous platelets were studied in healthy volunteers. Concentration-dependent functional alterations of isolated platelets were analyzed by flow cytometry, assessing changes in surface P-selectin expression and platelet-derived microparticles before and after in vitro stimulation with norepinephrine ranging from 10 nM to 100 μM. The thrombin receptor-activating peptide (TRAP) served as a positive control.ResultsDuring the first week following TBI, norepinephrine-mediated stimulation of isolated platelets was significantly reduced compared with volunteers (control). In the second week, the number of P-selectin- and microparticle-positive platelets was significantly decreased by 60% compared with the first week and compared with volunteers. This, however, was associated with a significantly increased susceptibility to norepinephrine-mediated stimulation, exceeding changes observed in volunteers and TBI patients during the first week. This pronounced norepinephrine-induced responsiveness coincided with increased arterio-jugular venous difference in platelets, reflecting intracerebral adherence and signs of cerebral deterioration reflected by elevated intracranial pressure and reduced jugular venous oxygen saturation.ConclusionClinically infused norepinephrine might influence platelets, possibly promoting microthrombosis formation. In vitro stimulation revealed a concentration- and time-dependent differential level of norepinephrine-mediated platelet activation, possibly reflecting changes in receptor expression and function. Whether norepinephrine should be avoided in the second post-traumatic week and whether norepinephrine-stimulated platelets might induce additional brain damage warrant further investigations.

Visualization of rat pial microcirculation using the novel orthogonal polarized spectral (OPS) imaging after brain injury

Recently, the novel optical system, orthogonal polarized spectral (OPS) imaging was developed to visualize microcirculation. Investigation of changes in microcirculation is essential for physiological, pathophysiological, and pharmacological studies. In the present study applicability of OPS imaging was assessed to study pial microcirculation in normal and traumatized rat brain. High quality images of rat pial microcirculation in normal and traumatized rats were generated with the OPS imaging, allowing to easily differentiate arterioles and venules with the dura remaining intact. In non-traumatized rats, mean vessel diameter of arterioles and venules of five different cortical regions was 19.1+/-2.7 and 22.2+/-1.4 microm, respectively. In the early phase following focal cortical contusion vessel diameter was significantly decreased in arterioles by 28% while diameter in venules was significantly increased by 27%. For technical reasons velocity in arterioles was not measurable. In venules, mean flow velocity of 0.68+/-0.08 mm/s was significantly decreased by 50% at 30 min after trauma. OPS imaging is an easy to use optical system allowing to generate high quality images and to reliably investigate pial microcirculation without having to remove the dura. This technique opens the possibility to perform longitudinal studies investigating changes in pial microcirculation.

Norepinephrine is Superior to Dopamine in Increasing Cortical Perfusion Following Controlled Cortical Impact Injury in Rats

Following traumatic brain injury catecholamines are routinely applied to increase cerebral perfusion. To date, it remains controversial if infusion of catecholamines is associated with diminished cerebral perfusion due to catecholamine-mediated vasoconstriction. The aims of the present study were to investigate the effects of norepinephrine and dopamine on cortical perfusion and brain edema following controlled cortical impact injury (CCII) in rats. Four hours after CCII, rats (n = 22) received either norepinephrine or dopamine with the aim of increasing MABP to 120 mm Hg for 90 minutes. Control rats were given NaCl. Cortical perfusion was measured before, during, and after catecholamine infusion using Laser Doppler flowmetry. Brain swelling was determined directly after the study period (8 hrs after CCII). Following CCII cortical perfusion was reduced by 40% compared to pre-trauma values in all rats. Parallel to the increases in MABP, cortical perfusion was significantly elevated under norepinephrine and dopamine, respectively (p < 0.05). Despite similar MABP values this increase was mostly sustained under norepinephrine. In control rats cortical perfusion remained diminished. Brain swelling was similar in all groups. Both norepinephrine and dopamine significantly increased cortical perfusion following CCII. Norepinephrine, however, was superior to dopamine in CBF. Based on increased CBF and unchanged brain swelling catecholamine-mediated vasoconstriction does not seem to occur under the present study design.

Norepinephrine-induced hyperglycemia does not increase cortical lactate in brain-injured rats

AbstractnObjective. Hyperglycemia aggravates ischemic brain damage. Since catecholamines increase hepatic gluconeogenesis, resulting in hyperglycemia, we investigated whether norepinephrine and dopamine elevate arterial blood glucose, thereby increasing pericontusional cortical glucose and lactate concentrations and brain edema in brain-injured rats.nDesign. Prospective, randomized, controlled animal study.nSubjects. Male Sprague Dawley rats.nInterventions. Physiological saline solution, norepinephrine, or dopamine were infused intravenously for 90xa0min beginning 4.5xa0h after inducing a focal cortical contusion. Blood glucose, lactate, and pericontusional cortical extracellular glucose and lactate were determined before, during and up to 60xa0min after the infusion period. Thereafter brains were removed to assess hemispheric water content.nMeasurements and results. Continuous norepinephrine and dopamine infusion significantly increased pericontusional glucose concentrations, being mostly sustained by norepinephrine (NaCl: 1.3±0.2, dopamine: 2.7±0.2, norepinephrine: 4.8±1.1xa0mM). While arterial blood glucose was only significantly elevated in norepinephrine-treated rats from 8.6±0.6 to 12.6±1.6xa0mM, the extracellular to blood glucose ratio was significantly increased in dopamine- and norepinephrine-treated rats (0.28±0.01 and 0.38±0.05 vs. 0.17±0.01). Plasma and pericontusional lactate remained unchanged, and brain edema was similar in all groups.nConclusions. Norepinephrine and dopamine significantly increased pericontusional glucose concentrations which did not elevate extracellular lactate and aggravate underlying posttraumatic edema formation. In addition to possibly increased facilitated endothelial glucose transport, the elevated extracellular to blood glucose ratio suggests a passive concentration- and pressure- dependent entry via a damaged blood-brain barrier. This might contribute to the observed reversible increase in extracellular glucose.

Temporal Profiles of Extracellular Nitric Oxide Metabolites Following Aneurysmal Subarachnoid Hemorrhage

The temporal profile of nitric oxide metabolite concentrations i.e. nitrite and nitrate (NOx) was investigated in brain parenchyma of patients following aneurysmal subarachnoid hemorrhage (SAH). In a subset of ten patients (7F/3M, age: 47 +/- 14 yrs) included in a prospective clinical trial on neurochemical intensive-care monitoring, microdialysis (MD) probes (CMA70, Sweden) were implanted at time of aneurysm surgery. Samples from patients clipped electively (n = 3) were considered normal in regard to SAH patients (n = 7). MD was performed for 162 +/- 63 hrs. NOx was measured off-line using a highly sensitive, fluorometric assay (2-3-diaminonaphtalene, DAN). NOx concentrations determined from electively operated patients averaged 36.7 +/- 9.6 microM (n = 59, pooled data). Regardless of the development of delayed ischemic neurological deficits (DIND), SAH patients showed a specific temporal profile of NOx consisting of an initial peak followed by an exponential decay. In detail, NOx decreased from initial values of 46.2 +/- 34.8 microM to 23.5 +/- 9.0 microM on day 6-7 after SAH (p < 0.05). Following SAH extracellular concentrations of NO metabolites decrease over time. This is in agreement with hypothetical NO scavenging by products of hemolysis. However, subsequent development of DIND cannot be explained by a lack of vasodilatory NO alone.

Differential concentration-dependent effects of prolonged norepinephrine infusion on intraparenchymal hemorrhage and cortical contusion in brain-injured rats.

Under clinical conditions catecholamines are infused to elevate cerebral perfusion pressure and improve impaired posttraumatic cerebral microcirculation. This, however, is associated with the risk of additional hemorrhage in the acute phase following traumatic brain injury. In the present study we investigated the dose-dependent effects of prolonged norepinephrine infusion on arterial blood pressure, blood glucose, and structural damage in brain-injured rats. At 4 h following induction of a focal cortical contusion (CCI), 40 rats were randomized to receive low (0.15), medium (0.3), or high dose (1 microg/kg/min) norepinephrine. Control rats were given equal volume of NaCl. Norepinephrine and NaCl were infused intravenously via Alzet osmotic pumps for 44 h. Mean arterial blood pressure (MABP), blood gases and blood glucose were determined before, at 4, 24, 48 h after CCI in repeatedly anesthetized rats (n = 28). Systolic arterial blood pressure (SABP) was measured using the tail cuff method in awake, restrained rats (n = 12). Cortical contusion and intraparenchymal hemorrhage volume were quantified at 48 h in all rats. MABP determined in anesthetized rats was only marginally increased. SABP was significantly elevated during infusion of medium and high dose norepinephrine in awake rats, exceeding 140 mm Hg. Medium and high dose norepinephrine significantly increased cortical hemorrhage by 157% and 142%, without increasing the cortical contusion volume. Low dose norepinephrine significantly reduced the cortical contusion by 44%. Norepinephrine aggravates the underlying brain damage during the acute posttraumatic phase. Future studies are needed to determine the least deleterious norepinephrine concentration.