Regulation of intracellular pH in single rat cortical neurons in vitro: a microspectrofluorometric study.

Intracellular pH (pHi) and the mechanisms of pHi regulation in cultured rat cortical neurons were studied with microspectrofluorometry and the pH-sensitive fluorophore 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. Steady-state pHi was 7.00 +/- 0.17 (mean +/- SD) and 7.09 +/- 0.14 in nominally HCO3(-)-free and HCO3(-)-containing solutions, respectively, and was dependent on extracellular Na+ and Cl-. Following an acid transient, induced by an NH1 prepulse or an increase in CO2 tension, pHi decreased and then rapidly returned to baseline, with an average net acid extrusion rate of 2.6 and 2.8 mmol/L/min, in nominally HCO3(-)-free and HCO3(-)-containing solutions, respectively. The recovery was completely blocked by removal of extracellular Na+ and was partially inhibited by amiloride or 5-N-methyl-N-isobutylamiloride. In most cells pHi recovery was completely blocked in the presence of harmaline. The recovery of pHi was not influenced by addition of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) or removal of Cl-. The rapid regulation of pHi seen following a transient alkalinization was not inhibited by amiloride or by removal of extracellular Na+, but was partially inhibited by DIDS and by removal of extracellular Cl-. The results are compatible with the presence of at least two different pHi-regulating mechanisms: an acid-extruding Na+/H+ antiporter, possibly consisting of different subtypes, and a passive Cl-/HCO3- exchanger, mediating loss of HCO3- from the cell.

Astrocytes fail to regulate intracellular pH at moderately reduced extracellular pH.

Microspectrofluorometry was used to study the regulation of intracellular pH (pHi) in 2'-7'-bis (carboxyethyl-)-5,6-carboxyfluorescein (BCECF)-loaded astrocytes. They rapidly regulated an acid transient induced by an NH4+ prepulse. This back regulation was blocked by removal of Na+, or by addition of amiloride, but was also inhibited when extracellular pH (pHe) was lowered. Furthermore, when cells were exposed to HEPES buffer with reduced or increased pHe, pHi changed in parallel. Thus, although the cells possess an efficient H+ extrusion mechanism they fail to regulate pHi to a normal value unless pHe is held constant. The results challenge the concept of a H+ regulatory site at the internal side of the exchanger regulating pHi to a constant value.

The influence of pH on glutamate- and depolarization-induced increases of intracellular calcium concentration in cortical neurons in primary culture.

The present experiments, carried out on neocortical neurons in primary culture with measurements of cytosolic calcium concentrations ([Ca2+]i) by microspectrofluorometric techniques, were designed to study how changes in extra- and intracellular pH (pHe and pHi, respectively) modulate the rise in [Ca2+]i due to glutamate exposure or potassium (K+)-induced depolarization. Although a reduction in pHe/pHi per se increased [Ca2+]i, the acidosis attenuated both the peak rise in [Ca2+]i following exposure to glutamate, and the plateau level observed during prolonged exposure. As a result, cells exposed to solutions with low pH consistently had lower [Ca2+]i values upon glutamate exposure than cells studied at normal pH. Alkalosis, i.e., an increase in pHe/pHi, had the opposite effect, accentuating the glutamate-induced [Ca2+]i transients. Experiments designed to separate changes due to extra- and intracellular pH suggested that the decisive event was the change in pHe. These results are consistent with the known effect of pHe on calcium flux through NMDA-gated ion channels. However, lowering of pHe had an equivalent effect on the rise in [Ca2+]i triggered by exposure of the cells to a K+ concentration of 50 mM. Thus, acidosis reduces influx of calcium through both agonist-operated and voltage-sensitive channels to such an extent that efflux/sequestration mechanisms suffice to maintain a lower [Ca2+]i.

Buffer capacity of rat cortical tissue as well as of cultured neurons and astrocytes.

The primary objective of this work was to assess the intrinsic nonbicarbonate buffer capacity (beta i) of cultured neurons and astrocytes and to compare the beta i values obtained to those of neocortical tissue. A second objective was to determine the pH dependence of beta i. Titration of homogenates of whole-brain cortical tissue and cultured neurons with NaOH and HCl gave beta i values of 25-30 mmol.l-1 x pH-1. The buffer capacity was essentially constant in the pH range of 6-7. Astrocytes showed a higher buffer capacity and a clear relationship between beta i and pH. However, beta i decreased when pH was reduced from 7 to 6. The beta i values derived from microspectrofluorometric studies on neurons and astrocytes were surprisingly variable, ranging from 10 to 50 mmol.l-1 x pH-1. The ammonia "step method" suggested that beta i increased dramatically when pH was lowered from 7 to 6 but the propionic "step method" failed to reveal such a pH dependence. Some techniques obviously give erroneous values for beta i, presumably because changes in buffer base concentration (due to transmembrane fluxes of H+, HCO3-, NH4+ or anions of weak acids) violate the principles upon which the calculations are based. From the results obtained by direct titration and with the propionate technique, we tentatively conclude that beta i in neurons and astrocytes are approximately 20 and 30 mmol.l-1 x pH-1, respectively. We further suggest that the term "intrinsic buffer capacity", as commonly used, is redefined.

The influence of calcium transients on intracellular pH in cortical neurons in primary culture.

The objective of this study was to assess the influence of Ca2+ influx on intracellular pH (pHi) of neocortical neurons in primary culture. Neurons were exposed to glutamate (100-500 microM) or KCl (50 mM), and pHi was recorded with microspectrofluorometric techniques. Additional experiments were carried out in which calcium influx was triggered by ionomycin (2 microM) or the calcium ionophore 4-Br-A23187 (2 microM). Glutamate exposure either caused no, or only a small decrease in pHi (delta pH approximately 0.06 units). When a decrease was observed, a rebound rise in pHi above control was observed upon termination of glutamate exposure. In about 20% of the cells, the acidification was more pronounced (delta pH approximately 0.20 units), but all these cells had high control pHi values, and showed gradual acidification. Exposure of cells to 50 mM KCl consistently increased pHi. Since this increase was similar in the presence and nominal absence of HCO3-, it probably did not reflect influx of HCO3- via a Na(+)-HCO3- symporter. Furthermore, since it occurred in the absence of external Ca2+ (or a measurable rise in Cai2+) it seemed independent of Ca2+ influx. It is tentatively concluded that the rise in pHi was due to reduced passive influx of H+ along the electrochemical gradient, which is reduced by depolarization. In Ca(2+)-containing solutions, depolarization led to a rebound increase in pHi above control. This, and the rebound found after glutamate transients, may reflect Ca(2+)-triggered phosphorylation and upregulation of the Na+/H+ antiporter which extrudes H+ from the cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in human intracerebral temperature in response to different methods of brain cooling.

The rectal, epidural, and intraventricular temperatures were continuously monitored in 10 seriously injured and unconscious patients admitted for neurosurgical intensive care. Different attempts were made to lower their brain temperatures. Isolated head cooling, whether with frozen liquid (Hypotherm Gel Kap; Flexoversal, Hilden, Germany) or a cooling helmet, had very limited effect. Nasopharyngeal cooling had no effect. During barbiturate coma, a considerable increase in brain temperature was observed. The administration of paracetamol was the single most effective method by which to lower brain temperature, at times by 2 degrees C and usually with a concomitant decrease in the temperature gradient between the rectum and the brain. However, in order to achieve a lasting reduction of brain temperature to 35 degrees C, we had to use a combination of head cooling and intensive whole-body cooling.

Intracerebral temperature in neurosurgical patients.

Recent laboratory results have indicated that the ischemic brain is very sensitive to minor variations in temperature. This has created new interest in hypothermia and brain temperature. There is, however, very little information available regarding human intracerebral temperature and its relation to body core temperature during normal and pathological circumstances. We therefore made continuous measurements of the temperature of the lateral ventricle in 15 neurosurgical patients utilizing a newly developed technique with copper-constantan thermocouples introduced through a plastic catheter also used for monitoring intracranial pressure. The intraventricular temperature was higher than the rectal temperature during approximately 90% of all measurements. The largest temperature gradient measured was 2.3 degrees C. Usually the difference between the temperature of the rectum and the brain was much smaller, the mean value being 0.33 degrees C. For the patients in the most severe condition, the rectal temperature was sufficiently close to the brain temperature to afford a reliable basis for adequate clinical judgment.

A method for monitoring intracerebral temperature in neurosurgical patients.

Current interest in brain temperature and selective brain cooling makes a method allowing for continuous monitoring of intracerebral temperature in humans desirable. The authors describe a safe, simple, and reliable technique using a thermocouple of copper and constantan in combination with intraventricular monitoring of intracranial pressure for measurement of brain temperature in neurosurgical patients.

Intracerebral microdialysis in clinical practice: baseline values for chemical markers during wakefulness, anesthesia, and neurosurgery.

OBJECTIVE: The study was undertaken to measure baseline values for chemical markers in human subjects during wakefulness, anesthesia, and neurosurgery, using intracerebral microdialysis. METHODS: Microdialysis catheters were inserted into normal posterior frontal cerebral cortex in nine patients who were undergoing surgery to treat benign lesions of the posterior fossa. The perfusion rate was 1.0 microl/min during anesthesia/neurosurgery and the early postoperative course and 0.3 microl/min during the later course. Bedside biochemical analyses of glucose, pyruvate, lactate, glycerol, glutamate, and urea were performed before, during, and after neurosurgery. After the bedside analyses, all samples were frozen for subsequent high-performance liquid chromatographic analyses of amino acids. RESULTS: The following baseline values were obtained during wakefulness (perfusion rate, 0.3 microl/min): glucose, 1.7+/-0.9 mmol/L; lactate, 2.9+/-0.9 mmol/L; pyruvate, 166+/-47 micromol/L; lactate/pyruvate ratio, 23+/-4; glycerol, 82+/-44 micromol/L; glutamate, 16+/-16 mmol/L; urea, 4.4+/-1.7 mmol/L. Marked increases in the levels of all chemical markers were observed at the beginning and end of anesthesia/surgery. CONCLUSION: The study provides human baseline levels for biochemical markers that can presently be measured at the bedside during neurointensive care. In addition, some changes that occurred under varying physiological conditions are described.

Human brain temperature during anesthesia for intracranial operations.

The intraventricular and rectal temperatures were registered in nine patients subjected to major surgery of the brain. Copper-constantan thermocouples were introduced into the lumen of an intraventricular catheter also used for perioperative monitoring of intracranial pressure. During anesthesia, the intraventricular temperature was higher than rectal temperature, the mean difference being 0.30 +/- 0.24 degrees C. No significant changes in intraventricular temperature were seen during different stages of the operations. It is concluded that during routine anesthesia rectal temperature can be relied on for a reasonable estimation of human brain temperature. It should be observed, however, that in the postoperative period, both rectal and intraventricular temperature rose considerably. In three patients, the intraventricular temperature rose as much as 2.5-4 degrees C, thus increasing the temperature gradient between rectum and brain. The relevance of these findings are discussed.

Intracerebral temperature in neurosurgical patients: intracerebral temperature gradients and relationships to consciousness level.

Information regarding human brain temperature is still rather sparse, which is surprising giving the no longer recent findings that the mammalian brain is very sensitive to minor variations in temperature. We therefore report the results of intracranial temperature monitoring in thirty neurosurgical patients. Intraventricular temperature was monitored with a thermocouple specifically designed for the purpose, and introduced through a plastic catheter used for monitoring of intracranial pressure, with simultaneous measurements of rectal and epidural temperature with commercially available thermocouples. Human intraventricular temperature is higher than central core temperature, and there is also a temperature gradient within the brain, with the central parts being warmer than the surface. The presence of an intracerebral temperature gradient was confirmed in two patients undergoing stereotactic thalatomy. We found no clear evidence for a correlation between consciousness level and brain temperature. Observations of brain temperature changes after clinical diagnosis of brain death are also reported.

Potential use of quantitative bedside CBF monitoring (Xe-CT) for decision making in neurosurgical intensive care.

During a 3-year period, mobile xenon-computerized tomography (Xe-CT) for bedside quantitative assessment of cerebral blood flow was used as an integrated tool for decision making during the care of complicated patients in our neurosurgical intensive care units (NSICU), in an attempt to make a preliminary evaluation regarding the usefulness of this method in routine work in the neurosurgical intensive care. With approximately 200 studies involving 75 patients, we identified six different categories where the use of bedside Xe-CT significantly influenced (or, with more experience, could have influenced) the decision making, or facilitated the handling of patients. These categories included identification of problems not apparent from other types of monitoring, avoidance of adverse effects from treatment, titration of standard treatments, evaluation of the vascular resistance reserve, assessment of adequate perfusion pressure and better utilization of resources from access to the bedside cerebral blood flow (CBF) technology. We conclude that quantitative bedside measurements of CBF could be an important addition to the diagnostic and monitoring arsenal of NSICU-tools.

Intracerebral microdialysis in neurosurgical intensive care patients utilising catheters with different molecular cut-off (20 and 100 kD).

OBJECTIVE: To compare the properties of a new intracerebral micro-dialysis catheter with a high cut-off membrane (molecular cut-off 100 kDalton) with a standard catheter (CMA70, molecular cut-off 20 kDalton). METHODS: Paired intracerebral microdialysis catheters were inserted in fifteen comatose patients treated in a neurosurgical intensive care unit following subarachnoid haemorrhage or traumatic brain injury. The high-cut-off catheter (D(100)) differed from the CMA 70 catheter by the length (20 mm) and cut-off properties of the catheter membranes (100 kDalton) and the perfusion fluids used (Ringer-Dextran 60). Samples were collected every 4-6 hours, analyzed bedside (for glucose, glutamate, glycerol, lactate, pyruvate and urea) and later in the laboratory (for total protein). RESULTS: Fluid recovery was similar for the two types of catheters, but significantly more protein was recovered by the D(100) catheter. The recovery of glycerol and pyruvate did not differ, while minor differences in recovery of glutamate and glucose were observed. The recovery of lactate was considerably lower in the D(100) catheter (p < 0.01), influencing the lactate/pyruvate-ratio. The patterns of concentration changes over time were consistent for all metabolites, and independent of type of catheter. CONCLUSION: Microdialysis catheters with high cut-off membranes can be used in routine clinical practice in the NSICU, adding the possibility of macro-molecule sampling from the extracellular space during monitoring.

Bedside monitoring of CBF with xenon-CT and a mobile scanner: a novel method in neurointensive care.

Combining previously independently established techniques our objective was to develop and evaluate a method for bedside qualitative assessment of cerebral blood flow in neurointensive care (NICU) patients. The CT-protocol was optimized using phantoms and comparing a mobile CT-scanner (Tomoscan-M, Philips) with two stationary CT scanners. Thirty-two per cent xenon was delivered with standard equipment (Enhancer 3000). Mean cortical flow in volunteers was 48 ml/min/100 g, with the mean vascular territorial flow varying between 45 and 66 ml/min/100 g. The potential clinical usefulness was illustrated in three patients with vasospasm following subarachnoid haemorrhage. Our conclusion is that quantitative bedside measurements of CBF can be repeatedly performed in an easy and safe way in a standard NICU-setting, using xenon-inhalation and a mobile CT-scanner. The method is useful for the decision-making, and is a good example of how the quality of multi-modality monitoring in the NICU can be developed and further diversified.

Monitoring of rectal, epidural, and intraventricular temperature in neurosurgical patients.

We will report our accumulated experience in monitoring of brain temperatures in neurosurgical patients. The intraventricular temperature was monitored with a thermocouple designed for the purpose. This thermocouple was introduced through a plastic catheter, which was also used for monitoring intracranial pressure. The rectal and epidural temperature was simultaneously measured, with commercially available thermocouples. Human brain temperature is higher than the central core temperature, and there is also a temperature gradient within the brain, with the central parts being warmer than the surface. The relationship between rectal, epidural and intraventricular temperatures is maintained during anaesthesia. We have also shown that it is possible to lower the temperature of the human brain.

Operations and re-operations for chronic subdural haematomas during a 25-year period in a well defined population.

In this retrospective study the hospital records of all patients being operated on for chronic subdural haematomas (CSD) at the Neurosurgical clinic in Lund in the years 1969, 1979, 1989, and 1993 were examined. 218 patients were operated on, 25 of whom had bilateral haematomas. During the 25-year period the incidence of surgically treated CSD rose from 2 to 5.3 per 100,000 inhabitants per year. The mean age (70.5 years) and the relationship males: females (2:1) did not significantly change. The clinical condition of the patients on admission steadily improved during the period. The relative proportion of patients with known chronic alcoholism decreased over the years, but the proportion of patients suffering from other complicating diseases increased, as did the proportion of patients treated with anticoagulants. There was no mortality directly related to surgery, but if defined as deaths within one month after surgery, the overall mortality rate was 3.2%. 84.2% of the patients improved following the first operation, in a majority of cases back to the premorbid state. The relative frequency of re-operations for CSD was 12.3% and did not significantly change during the period. No pre- or peri-operative variable could be identified which could predict who of the patients was at higher risk of re-operation. Surprisingly, the data suggest that the less experienced neurosurgeons had better operative results compared with their older collegues.