The square-wave approach to impedance measurement of brain tissue water dynamics.

Electrical properties of living soft tissue have been used to analyze their structure and function. Presently, the 'admittance locus' method, with the sine-wave signal of changing frequency, is the most informative continuous method for analyzing extra-and intracellular water content in brain tissue. Using the square-wave signal in lieu of the sine-wave signal, we can avoid cumbersome and costly measurements and facilitate real-time data processing. An isolation-calibration device was developed for the present study in order to condition and stabilize electrical current through the brain cortex. This device was also used for impedance calibration before and after the experiments. We propose a simple algorithm for data analysis on the basis of equivalent circuit approach, which allows to develop a computer program for data processing. Preliminary experiments on rat brains were carried out with a 0.2-0.5 mm stainless-steel tetrapolar electrode system. These studies showed good linearity between stimulating currents (I = 5-30 microA) through the external electrodes in the brain cortex and a drop in voltage which was measured by 2 inner electrodes. The results of the device and the program accuracy tests allow us to choose the optimal range for the working current. We can recommend this method for usage in animal experiments.

Traumatic injury induces interleukin-6 production by human astrocytes.

The brain is being evaluated as a de novo source of cytokines. Because recent evidence indicates that interleukin-6 (IL-6) may influence blood-brain barrier function and vascular permeability, we have sought to determine whether mechanical injury can directly induce in situ cerebral IL-6 production. Adult human astrocyte cultures were subjected to mechanical injury by the in vitro method of fluid percussion barotrauma, developed in our laboratory. Serial supernatant samples were collected for 8 h and evaluated for IL-6 activity using a proliferation assay employing the dependent B cell hybridoma cell line, B9. At optimum injury, the IL-6 level became significantly (P < 0.0001, analysis of variance) elevated from baseline 2 h after trauma and continued to increase over the observation period. Our study shows that following mechanical injury human astrocytes produce IL-6, which may contribute to post-traumatic cerebrovascular dysfunction. Elucidating the precise role of intracerebral cytokines is essential to our understanding of the mechanism responsible for post-traumatic cerebrovascular dysfunction.

A square signal wave method for measurement of brain extra- and intracellular water content.

Brain tissue electrical impedance is a commonly used method to evaluate the dynamics of brain edema. We have found the square wave impedance method simpler and more cost-effective than the currently used sine wave impedance method. This square wave method avoids the necessity for expensive frequency control and amplitude-phase measuring devices as well as simplifying on-line data processing. In our experiments the electrical impulse was generated by a pulse generator of Macintosh data acquisition system. The signal (I = 11 muA, t = 2-20 ms) was delivered every 2-3 s external electrodes of a tetrapolar system through a specially designed isolation-calibration device. This electrode system was inserted into the cerebral cortex of experimental animals (rat). The cerebral cortex was found to have linear electrical properties in the 5-30 muA range. Our impedance measurement system was tested in calibration trials, and showed system reliability and accuracy. The system was also tested in pilot experiments, in vivo, in a rat brain osmotic edema model.

Traumatic brain injury, hemorrhagic shock, and fluid resuscitation: effects on intracranial pressure and brain compliance.

Intracranial hypertension following traumatic brain injury is associated with considerable morbidity and mortality. Hemorrhagic hypovolemia commonly coexists with head injury in this population of patients. Therapy directed at correcting hypovolemic shock includes vigorous volume expansion with crystalloid solutions. It is hypothesized that, following traumatic brain injury, cerebrovascular dysfunction results in rapid loss of brain compliance, resulting in increased sensitivity to cerebrovascular venous pressure. Increased central venous pressure (CVP) occurring with vigorous crystalloid resuscitation may therefore contribute to the loss of brain compliance and the development of intracranial hypertension. The authors tested this hypothesis in miniature swine subjected to traumatic brain injury, hemorrhage, and resuscitation. Elevated CVP following resuscitation from hemorrhage to a high CVP significantly worsened intracranial hypertension in animals with concurrent traumatic brain injury, as compared to animals subjected to traumatic brain injury alone (mean +/- standard error of the mean: 33.0 +/- 2.0 vs. 20.0 +/- 2.0 mm Hg, p < 0.05) or to animals subjected to the combination of traumatic brain injury, hemorrhage, and resuscitation to a low CVP (33.0 +/- 2.0 vs. 24.0 +/- 2.0 mm Hg, p < 0.05). These data support the hypothesis that reduction in brain compliance can occur secondary to elevation of CVP following resuscitation from hemorrhagic shock. This may worsen intracranial hypertension in patients with traumatic brain injury and hemorrhagic shock.

Contribution of increased cerebral blood volume to posttraumatic intracranial hypertension.

Cerebrovascular dysfunction following acute brain injury (BI) may be the critical mediator of excess morbidity and mortality after BI. Despite aggressive therapy, death often is caused by refractory intracranial hypertension (IH). An understanding of the contributions of cerebrospinal fluid (CSF) and vascular factors to IH after BI is essential for management of intracranial pressure (ICP). Marmarou et al. showed that CSF accounted for only one third of the ICP rise after BI. We hypothesized that a vascular mechanism is predominant. Cerebral cortical reflectance photoplethysmography (IP) and radioactively labeled red blood cells were employed to study cerebral blood volume (CBV) changes associated with increased ICP after BI in miniature swine. Immediate posttraumatic IH could be attributed almost entirely to increased CBV. An early elevation in ICP immediately after BI (t = 0) was accompanied by a large increase in CBV compared with pre-BI levels (19.2 +/- 4.9 vs. 8.9 +/- 2.7 mL/100 g tissue, p < 0.05). Decreased CBV corresponded to lower ICP within 1 hour, followed by a slow rise that paralleled the increase in ICP. The CBV (16.1 +/- 3.3 vs. 8.9 +/- 2.7, p < 0.05) and ICP (23 +/- 2.2 vs. 9 +/- 0.6, p < 0.05) were higher at 6 hours than at baseline. Based on compartmental analysis, the data indicate that ICP changes immediately after BI and within 6 hours are predominantly caused by increased CBV.

Human astrocyte production of tumour necrosis factor-alpha, interleukin-1 beta, and interleukin-6 following exposure to lipopolysaccharide endotoxin.

The cytokines, tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, and IL-6, have been found in the human central nervous system. Recent studies have demonstrated that murine astrocytes produce these cytokines when induced with lipopolysaccharide endotoxin (LPS). The present study investigates the kinetics of TNF-alpha, IL-1 beta, and IL-6 production by normal adult human astrocytes when exposed to LPS.

Alterations in intracranial pressure and cerebral blood volume in endotoxemia.

Marked deterioration of neurologic function accompanies organ dysfunction in systemic sepsis. Although previous hypotheses have suggested that cerebral hypoperfusion, anoxia or progressive edema of the brain may be causative, the pathogenesis remains unknown. Patients with sepsis with stable or supported hemodynamics and adequate oxygenation may manifest confusion, stupor or coma. Recent evidence has demonstrated that the brain is the source of many classical mediators of inflammation after various forms of injury. These mediators, including the leukotrienes, have pronounced effect on cerebrovascular function. Endotoxin is known to stimulate the release of arachidonate from cell membranes, the rate limiting step in leukotriene synthesis. The current studies were performed to test the hypothesis that neurologic dysfunction associated with endotoxemia is characterized by alterations in cerebrovascular permeability or vasomotor function manifested by intracranial hypertension, or both. We studied the response of miniature swine to experimental endotoxemic shock and compared this response with hemorrhagic hypotension. We observed a dramatic elevation of intracranial pressure in swine subjected to endotoxemic shock, despite arterial hypotension. Moreover, estimation of cerebral blood volume (CBV) by reflectance infrared photoplethysmography demonstrated a dramatic increase in CBV, which corresponded to this elevation in intracranial pressure. However, cerebral cortical oxygen saturation was significantly reduced despite this net increase in CBV, indicative of an increase in the venous volume of the brain, while arterial volume remained the same or decreased from baseline levels. Oxygen extraction across the brain decreased during this same period compared with baseline and control values. These results demonstrate that endotoxemia is associated with the development of intracranial hypertension and an increase in CBV secondary to elevation of cerebrovascular venous volume coupled with reduced oxygen extraction across the brain. This evidence of cerebrovascular dysfunction probably represents blood flow maldistribution, similar to that seen in other organs with sepsis, suggesting a cause for altered neurologic function in systemic sepsis.

Fluid percussion barotrauma chamber: a new in vitro model for traumatic brain injury.

Advances in the understanding of the pathophysiology of traumatic brain injury have implicated a number of cellular events as fundamental to the evolution of neurologic dysfunction in this process. Following the primary biomechanical insult, a highly complex series of biochemical changes occur, some of which are reversible. The development of fluid percussion injury as an in vivo model for traumatic brain injury has greatly improved our ability to study this disease. However, a comparable in vitro model of biomechanical injury which would enable investigators to study the response to injury in isolated cell types has not been described. We have developed a model of transient barotrauma in cell culture to examine the effects of this form of injury on cell metabolism. This model employs the same fluid percussion device commonly used in in vivo brain injury studies. The effect of this injury was evaluated in monolayers of human glial cells. Cell viability by trypan blue exclusion and the production of leukotrienes following increasing barotrauma was investigated. This model provided a reproducible method of subjecting cells in culture to forces similar to those currently used in animal experimental head injury.

Leukotriene production by human glia.

Elevated intracranial pressure and acute cerebrovascular changes following head injury remain the principle challenge in the management of traumatic brain injury. Recent work has demonstrated that leukotrienes can induce increases in blood brain barrier permeability and alter cerebrovascular dynamics. We investigated whether human astroglia in culture: 1. generate specific leukotrienes; 2. how they metabolize leukotrienes, and; 3. if astroglia generate leukotrienes in response to barotraumatic injury. Human astroglial cultures established from normal human brain obtained at surgery were exposed to either ionophore, exogenous 3H-LTC4, or barotraumatic injury. Supernatants were assayed for specific leukotrienes by one of three methods: HPLC, radioimmunoassay, or enzyme-immunoassay. Glial cells exposed to exogenous LTC4 metabolized nearly all of the LTC4 to LTD4 and LTE4 within 20 minutes. Glial cells stimulated with ionophore produced mostly LTC4 at five minutes after stimulation and LTD4 and LTE4 at fifteen minutes after stimulation. Glial cells subject to barotraumatic injury produced LTC4 in concentrations of 40-200 pg/ml 15 minutes after injury. These results demonstrate that human astroglial cells are capable of rapidly generating and degrading LTC4 and this capability of glial cells may play an important role in the pathophysiology of cerebrovascular changes following head injury.

Human glial cell production of lipoxygenase-generated eicosanoids: a potential role in the pathophysiology of vascular changes following traumatic brain injury.

Acute cerebrovascular changes which occur following traumatic brain injury represent a highly complex, multifactorial pathophysiologic process which is poorly understood. It is now recognized that, under normal conditions, the brain is a source of a variety of arachidonic acid metabolites which are synthesized by both cyclooxygenase and lipoxygenase. The specific cellular source of these highly vasoactive substances remains controversial. Recent work has demonstrated that lipoxygenase products were detected by immunosensitive assay in whole brain samples from a gerbil concussive injury model, yet the production of leukotrienes could not be accounted for by cerebral vessels and their contents alone. It has been theorized that the probable source for these metabolites is the cortical neuron. We sought to elucidate whether cultured human glial cells, obtained from specimens removed at the time of surgery, are a significant source of lipoxygenase products as measured by high performance liquid chromatography (HPLC). We observed that these cells consistently produced 5, 12, and 15-HETE class eicosanoids despite failure to produce significant cyclooxygenase products. These preliminary findings are of considerable interest because these lipoxygenase products are known to be highly vasoactive as well as potent mediators of increased vascular permeability. Since it is known that mechanical perturbation of cell membranes stimulates the release of arachidonic acid from membrane phospholipids, it is conceivable that the production of these eicosanoids following traumatic brain injury could account for local cerebrovascular changes including both vasospasm and interstitial edema formation.

Regional acetylcholine metabolism in brain during acute hypoglycemia and recovery.

Insulin-induced hypoglycemia in normothermic rats caused progressive neurological depression and differentially altered regional cerebral acetylcholine metabolism. Reductions of plasma glucose from 7.7 mM (control) to 2.5-1.7 mM (moderate hypoglycemia associated with decreased motor activity) or 1.5 mM (severe hypoglycemia with lethargy progressing to stupor) decreased glucose concentrations in the cerebral cortex, striatum, and hippocampus to less than 10% of control. Moderate hypoglycemia diminished acetylcholine concentrations in cortex and striatum (21% and 45%, respectively) and reduced [1-2H2, 2-2H2]choline incorporation into acetylcholine (62% and 41%, respectively). Severe hypoglycemia did not reduce the acetylcholine concentration or synthesis in cortex and striatum further. The concentrations of choline rose in the cortex (+53%) and striatum (+130%) of animals that became stuporous but a similar rise in [1-2H2, 2-2H2]choline left the specific activities of choline in these structures unchanged. Even severe hypoglycemia did not alter the hippocampal cholinergic system. In rats that developed hypoglycemic stupor and were then treated with glucose, the animals recovered apparently normal behavior, and the concentrations of acetylcholine and the incorporation of [1-2H2, 2-2H2]-choline into acetylcholine returned to control values in the striatum but not in the cerebral cortex. Thus, impaired acetylcholine metabolism in selected regions of the brain may contribute to the early symptoms of neurological dysfunction in hypoglycemia.