Herniated lumbar disc material as a source of free glutamate available to affect pain signals through the dorsal root ganglion.

STUDY DESIGN: Combined prospective human cohort and prospective controlled animal model. OBJECTIVES: To determine whether free glutamate is available in herniated disc material in concentrations sufficient to diffuse to glutamate receptors and affect the activity of neurons in the dorsal root ganglion that may transmit pain information. SUMMARY OF BACKGROUND DATA: The severity of lumbar radicular pain cannot be fully explained by physical pressure on nerve roots or ganglions. In experimental models, inflammatory processes are relatively modest under conditions of disc herniation. The hypothesis for the current study was that the proteoglycan link and core proteins, which contain high fractions of acidic amino acids, may be a source of glutamate when enzymatically degraded in an environment without glutamate reuptake systems. Glutamate would be free to diffuse to the dorsal root ganglion to affect glutamate receptors. METHODS: Disc material was harvested during surgery from herniated and nonherniated portions in patients undergoing elective lumbar disc surgery and subjected to immunohistochemistry and high-performance liquid chromatography for assessment of the presence of extracellular disc matrix glutamate. Miniosmotic pumps with differing concentrations of radiolabeled glutamate based on human data were implanted in the rat epidural space for 72 hours and dorsal root ganglion (DRG) in the region were harvested. RESULTS: Densitometry of disc matrix demonstrated immunohistochemical evidence for significant extracellular glutamate (P < 0.002). High performance liquid chromatography showed significant concentrations of glutamate in disc material and significantly more in herniated than in nonherniated disc material (P < 0.05). Significant radiolabeling of the dorsal root ganglion after epidural glutamate infusion was found at concentrations two orders of magnitude below measured disc glutamate levels. Autoradiography demonstrated radiolabeling of adjacent DRG. CONCLUSIONS: Glutamate originating from degenerated disc proteoglycan may diffuse to the dorsal root ganglion and effect glutamate receptors. Consideration may be given to treating disc radiculopathy with epidural glutamate receptor antagonists.

Effect of hypoxia on psychomotor performance during graded exercise.

We evaluated the combined effects of reduced O2 (12%) and graded ergometer exercise (10, 70, and 140 W) on the performance of a psychomotor task. Six men participated in two test sessions each. Each session began with the baseline data (air) and finished with exposure data (12% or 21%, random, unidentified). The gas mixtures were air (control condition) and 12% O2-balance N2 (12% O2 condition). The psychomotor task score was degraded during the 140 W work rate of the 12% O2 condition (p less than 0.05). Reductions of the SaO2 (p less than 0.05) at each work rate proved that hypoxia existed during exercise in the 12% O2 condition. The arterial blood gases indicated that respiratory alkalosis was mixed with metabolic acidosis during the hypoxic exercise. The results differ from previously reported findings of an arousal effect of exercise on psychomotor performance and the decrement of task performance at work rates less than or equal to 50 W. We conclude that hypoxia degraded the performance of the psychomotor task during graded exercise.

Sensitivity of cultured mammalian cells to oxidative stress: adaptation to repeated exposures of hyperbaric oxygen.

Four established cell lines (mouse neuroblastoma, N2A; Chinese hamster lung, V79; Chinese hamster ovary, CHO; and rabbit kidney, RK13) were made O2-tolerant by repetitive exposure to hyperbaric oxygen (HBO). The cultures were exposed to 100% O2 at pressures ranging from 6 to 10 ATA for time periods up to 3 h, and the surviving cells were regrown to monolayer confluency and reexposed; by the end of three cycles of treatment these cells were tolerant to exposures of 10 ATA O2 for greater than 5 h. The development of O2 tolerance was measured by enzyme and morphologic indices. Results showed that all of the cell lines tested could be made O2 resistant. However, qualitative differences were found. RK13 cells were more resistant to HBO than the other cell types tested. The technique provides a powerful adjunct to current methods that study the effects of oxidative stress in mammalian cells. The ability to generate O2-resistant cells in only 3-4 wk provides a considerable time savings over published efforts (12-20 mo.). In addition, rapid screening of various cell lines may lead to discovery of O2-resistant cell types which will provide insight into the factors inherent in the development of oxygen tolerance.

Symptomatology during hypoxic exposure to flame-retardant chamber atmospheres.

Hypoxia was studied in 12 men during 63-h exposures to 17 and 13% O2, with the subjects serving as their own controls by repeating the measurements in 21% O2. All test atmospheres were contaminated with 0.9% CO2 to simulate the condition of living aboard submarines. The mean SaO2's were 97-98% in all conditions of 21% O2, 96% in 17% O2 (n.s.), and 92% in 13% O2 (P less than 0.05). The blood concentrations of 2,3-diphosphoglycerate were elevated in 13 and 17% O2 (P less than 0.05). Seventeen percent O2 did not cause significant symptoms of environmental stress; however, 13% O2 caused symptoms of acute mountain sickness in 5 of 12 men. In the last 7 h of exposure to 17% O2, reduction of the barometric pressure to 576 Torr reduced the ambient PO2 to 98 Torr (similar to the PO2 of 13% O2 at normobaric pressure). This induced symptoms of acute mountain sickness in 3 of 11 men. All symptomatology and physiologic changes were reversed during recovery in 21% O2. Monitoring devices indicated the presence of volatile organic contaminants at a mean concentration of 6.1 ppm in the chamber atmosphere. Combustion tests in the occupied chamber showed that flame propagation was retarded by lowering the O2 concentration from 21 to 13-17%. We conclude that men can live comfortably in a normobaric, flame-retardant atmosphere consisting of 17% O2-0.9% CO2-6.1 ppm volatile organic compounds-balance N2.

Progression of and recovery from pulmonary oxygen toxicity in humans exposed to 5 ATA air.

Animal studies suggest that pulmonary oxygen toxicity proceeds more slowly in diluted oxygen breathing mixtures than in pure oxygen at the same inspired partial pressure. We exposed 12 healthy subjects to air at 5 ATA (PiO2 = 1.05 ATA) in a hyperbaric chamber for 48 h, and compared the rate of development of symptoms of O2 toxicity to rates seen in previous studies using 100% O2 at 1 ATA. Symptoms consisted of chest tightness, cough, substernal discomfort, exertional dyspnea, anorexia, nausea and vomiting, headache and digital paresthesias starting at about 12 h, and continuing several days into the recovery period. Pulmonary function changes consisted of significant decrements in vital capacity, flow rates, and DLCO. Initial recovery was in a 0.50 ATA oxygen atmosphere, with the majority of subjects showing definite recovery in both symptoms and pulmonary function. Subjects showed complete recovery in about 8 d, although symptoms of fatigue and exertional dyspnea continued for a month in some cases. In contrast, none of the above changes were noted in an additional 6 subjects exposed to a 5 ATA environment with 6% oxygen (PiO2 = 0.30 ATA). No change in resting gas exchange, as indicated by alveolar-arterial oxygen gradients, was detected in either group. Comparison of these data to that for pure oxygen studies reveals no significant difference in the progression or character of pulmonary oxygen toxicity.

Phasic changes in bone CO2 fractions, calcium, and phosphorus during chronic hypercapnia.

The bone CO2 buffering system and bone calcium and phosphorus were studied in guinea pigs exposed to 1% CO2 for periods up to 8 wk and killed at weekly intervals together with control animals of the same age. Measurements were made of arterial CO2 tension, pH, standard bicarbonate, and bone Ca and P. Heat-stabile bone CO2 (carbonate) was determined as dry bone CO2 and heat-labile bone CO2 (bicarbonate) as delta wet-dry bone CO2. During the first 3-4 wk of exposure to 1% CO2, a systemic acidosis was found as indicated in a lowered pH, increased arterial CO2 tension, and decreased standard bicarbonate. The acidosis subsided during the last 4 wk of exposure. Phasic changes in bone bicarbonate were observed as shown in immediate rise lasting for 2 wk followed by a 2-wk decline and second rise after 6 and 8 wk. Bone carbonate exhibited the opposite change during the first 4 wk and thereafter remained stable at an elevated level. Bone Ca and P fell in association with increasing bone bicarbonate and rose with increasing bone carbonate.

Bone CO2-titration curves in acute hypercapnia obtained with a modified titration technique.

Bone CO2-titration curves were obtained in mature rats weighing 500-600 g. Animals were exposed for one hour to 1, 3, 5, 10, or 15% CO2 in air. Measurements of bone CO2, were made using a modified titrimetric analysis on fresh and oven-dried samples of paired rat femurs. A manometric method was used for comparison. Arterial blood samples were obtained for measurements of partial pressure of CO2 in arterial blood (PaCO2). Within the range of environmental CO2 concentrations studied, a linear relationship was observed between the PaCO2 and the increment in fresh bone CO2 content. This relationship is defined by the equation: delta fresh bone CO2 (mmol/kg) = 61.8 +/- 0.68 PaCO2. The CO2 increment was confined to a heat-labile, presumably soluble pool comprising 10.5% of the total bone CO2 content. No change in the water content of the bone was observed as a result of acute CO2 exposure. The results of this study demonstrate the rapid in vivo CO2 uptake of bone in response to exposure to increased CO2 levels.

CO2-induced kidney calcification.

Light microscopic examination of kidney tissue of guinea pigs exposed to 1.5% CO2, 21% O2, and balance N2 for periods as long as 42 days and of rats exposed to the same CO2 concentrations for up to 91 days showed that the incidence of focal kidney calcification increased with length of exposure. Calcification occurred primarily in the tubules of the renal cortex. Another group of guinea pigs were exposed to 1% CO2, 21% O2, and the balance N2 for periods up to six weeks and were later killed at regular intervals, together with control animals of the same litter. In the exposed animals, arterial PCO2 was elevated by 3-4 mmHg and hydrogen ions by about 4 nmol/liter. The standard bicarbonate level was lowered by 1-1.5 mmol, indicating a lack of renal reabsorption of bicarbonate (HCO3), which in turn placed greater stress on the bone buffer system and apparently caused bone calcium and phosphorus mobilization. Bone calcium and phosphorus levels exhibited a cyclic decrease, which resulted in cyclic hypercalcemia and hyperphosphatemia, after one week and six weeks of exposure to 1% CO2. Kidney calcium content increased significantly after two weeks of exposure (27%) and remained at this elevated level during subsequent exposures between the third and sixth weeks. These findings indicate that once the kidney calcification process has started, kidney mineralization is independent of fluctuations in the blood calcium level. A rise in plasma phosphate level that occurred after one day of exposure could have been a precipitating factor in the calcification process. The small but consistent increases in ionized calcium during a 4-week exposure to 1% CO2 may have stimulated the parathyroid, causing an increased blood calcium level that was independent of the two calcium tides in the blood associated with marked bone calcium loss.

Effect of intermittent exposure to 3% CO2 on respiration, acid-base balance, and calcium-phosphorus metabolism.

One subject was exposed for six days to increasing levels of CO2, rising at a constant rate from 0.03 to 3.0% CO2 within a 15-h period followed by 9 h of air breathing. To assess acid-base parameters, arterialized capillary blood was taken from a finger twice daily (at 8 a.m. and 11 p.m.) at times corresponding to the beginning and end of the intermittent exposure to CO2. Venous blood samples were obtained on alternate days at the same times. Urine specimens were collected twice daily. The subject was on a liquid diet. Resting respiratory minute volume (VE), oxygen consumption (VO2), carbon dioxide excretion (VCO2), alveolar carbon dioxide and oxygen tension (PACO2) and PAO2) were measured twice daily. PACO2 and PAO2 were also determined at the end of breath-holding twice daily; CO2 tolerance tests and lung function tests were also carried out. In contrast to the effects of chronic exposure to 3% CO2, the CO2 tolerance tests showed an increased sensitivity (increase of slope) and breath-holding PACO2 did not change, indicating that acclimatization to CO2 did not develop. The ventilatory response to CO2 was not sufficient to prevent CO2 accumulation in the body; this accumulation was eliminated during the nightly air-breathing periods on the fourth and fifth days, indicated by higher values of PaCO2 and PACO2. The known renal response to hypercapnia, consisting of an increased excretion of titratable acidity, ammonia, and hydrogen ion excretion, occurred but was interrupted after the first day and was triggered again on the fourth and fith days when accumulated CO2 was released from body CO2 stores. The second renal response was associated with a marked calcium excretion, which suggests that bone CO2 stores were involved.

Proliferation of pneumocyte II cells in prolonged exposure to 1% CO2.

Guinea pigs were exposed to 1% CO2 in a mixture of 21% O2, balance N2 and were killed at weekly intervals, together with control animals housed in the same type of environmental chamber and exposed to normal ambient CO2. The exposed animals showed a persistent elevation of PaCO2, averaging about 4 mmHg, and a small decrease in pH (-0.04 units). During the whole exposure period standard bicarbonate remained 1-1.5 mEq below control levels, indicating a failure of the kidney to increase bicarbonate reabsorption. Electron microscopic studies after 4 and 6 weeks of exposure to 1% CO2 showed ultrastructural changes of the lungs, consisting of marked increases in the size and number of pneumocyte II cells that were still present two weeks and to a lesser extent four weeks after recovery. Changes in the pneumocyte II cell were postulated to be compensatory reactions to impairing CO2 effects on the alveolar lining cell (Type I cell).

Effect of prolonged exposure to 0.5% CO2 on kidney calcification and ultrastructure of lungs.

Guinea pigs were exposed for up to 8 weeks to 0.5% CO2, 21% O2, and balance N2. Control groups of the same age were kept simultaneously in environmental chambers on air. A slight increase in PaCO2 and decrease in pH were observed at various periods of exposure to 0.5% CO2. After eight weeks of exposure, an increased kidney calcification, indicated by increased kidney calcium content, was found. Plasma calcium was significantly elevated at this point, apparently because of the release of calcium from bone. After 8 weeks of recovery on air that followed 8 weeks of exposure to 0.5% CO2, values had returned to control levels. No significant ultrastructural changes were found in the lungs after 4, 6, and 8 weeks of exposure to 0.5% CO2.

Calcium, magnesium, and phosphorus metabolism, and parathyroid-calcitonin function during prolonged exposure to elevated CO2 concentrations on submarines.

Studies of calcium and phosphorus metabolism and acid-base balance were carried out on three Fleet Ballistic Missile (FBM) submarines during prolonged exposure to elevated concentrations of CO2. The average CO2 concentration in the submarine atmosphere during patrols ranged from 0.85% to 1% CO2. In the three studies, in which 9--15 subjects participated, the urinary excretion of calcium and phosphate fell during the first three weeks to a level commensurate with a decrease in plasma calcium and increase in phosphorus. In the fourth week of one patrol, a marked increase was found in urinary calcium excretion, associated with a rise in blood PCO2 and bicarbonate. Urinary calcium excretion decreased again during the 5th to 8th week, with a secondary decrease in blood pH and plasma calcium. During the third patrol, the time course of acid-base changes corresponded well with that found during the second patrol. There was a trend toward an increase in plasma calcium between the fourth and fifth week commensurate with the transient rise in pH and bicarbonate. Plasma parathyroid and calcitonin hormone activities were measured in two patrols and no significant changes were found. Hydroxyproline excretion decreased in the three-week study and remained unchanged in the second patrol, which lasted 57 days. It is suggested that during prolonged exposure to low levels of CO2 (up to 1% CO2), calcium metabolism is controlled by the uptake and release of CO2 in the bones. The resulting phases in bone buffering, rather than renal regulation, determine acid-base balance.

First apparent dissociation constant of carbonic acid, pK'1, of plasma and erythrocytes.

The first apparent dissociation constant of carbonic acid, pK'1, of plasma and red cells was determined on venous blood of ten healthy, adult, male, human subjects. pH and PCO2 of plasma and red cells were analyzed electrometrically and a micromanometric method was used for the determination of total carbon dioxide content. Erythrocyte carbamino hemoglobin levels were estimated and used for the correction of erythrocyte pK'1. Each blood sample was subjected to the following regimen before centrifugation, 1) As drawn from the antecubital vein, 2) Oxygenated with a 5% CO2, O2 balance gas mixture, and 3) Reduced with a 5% CO2, N2 balance gas mixture. pK'1 of plasma and red cells are presented: (see article). The consistently larger values for red cell pK'1 than the respective plasma data may be attributed to the greater amount of carbamino hemoglobin concentration present in the erythrocytes. A simplified method for the calculation of erythrocyte bicarbonate concentration using the experimentally determined red cell pK'1 value has been formulated. The method involves the use of a regression equation relating plasma and red cell pH, the equivalence of plasma and red cell PCO2, along with the experimentally determined red cell pK'1.

Effect of chronic hypercapnia on body temperature regulation.

Guinea pigs and rats exposed to 15% CO2 for 7 days showed a parallel time course of changes in pH, body temperature (TB), and oxygen consumption (VO2). Between 1 and 6 h of exposure the maximal drop in actual pH occurred in guinea pigs simultaneously with the maximal fall in TB and VO2. During the subsequent period pH TB, VO2 rose again. Skin blood content (heat loss) also exhibited a biphasic pH-dependent time course. Animals showing no partial compensation of respiratory acidosis during 3 days exposure also failed in raising their TB back to normal in this time. The behavior of TB was found to be a good indicator of the acid-base status and adaptive potential of the animals to hypercapnia. Similar results were obtained in rats. Thermo-regulatory processes in the hypothalamus were affected during exposure to 15% CO2. Both guinea pigs and rats showed a decrease in norepinephrine content of the hypothalamus during the first part of exposure reaching a maximal fall at the end of 24 h. The serotonin content increased slightly during this period. During prolonged exposure to 3% CO2 for 7 days, TB showed a transient rise, and VO2 was slightly elevated.