Mannitol and furosemide are used to reduce increased intracranial pressure (ICP) and to reduce brain bulk during neurosurgery. One mechanism by which these changes might occur is via a reduction in brain water content. Although mannitol and furosemide are commonly used in combination, there has been no formal evaluation of the interactive effects of these two drugs on brain water. The effect of mannitol and furosemide alone and in combination on water content of normal rat brain was examined.
The lungs of rats anesthetized with halothane were mechanically ventilated to maintain normal physiologic parameters. After baseline measurement of plasma osmolality, mannitol (1, 4, or 8 g/kg), furosemide (2, 4, or 8 mg/kg), or a combination of furosemide (8 mg/kg) and mannitol (1, 4, or 8 g/kg) was administered intravenously over approximately 15 min. One hour later, plasma osmolality was measured, the animals were killed, and brain water content was determined by wet and dry weight measurements.
Mannitol produced a dose-dependent increase in plasma osmolality and reduction of brain water content. There was a linear relation between plasma osmolality and brain water content. Furosemide alone did not affect plasma osmolality or brain water at any dose. The combination of furosemide with mannitol resulted in a greater increase in plasma osmolality than seen with mannitol alone and a greater decrease in brain water at 4 and 8 g/kg of mannitol.
The doses of mannitol and furosemide utilized were much larger than clinically applicable doses and were selected to maximize the ability to detect effect on brain water. The combination of mannitol and furosemide resulted in greater reduction of brain water content than did mannitol alone. Furosemide enhanced the effect of mannitol on plasma osmolality, resulting in a greater reduction of brain water content. Potential interaction (if any) of smaller, clinically used doses of mannitol and furosemide cannot be surmised from the current study.
DIURETICS have long been used in the management of increased intracranial pressure (ICP). Osmotic diuretics such as mannitol consistently reduce increased ICP. 1–6The effect of loop diuretics such as furosemide has been less consistent, with ICP reductions reported by some 2–4,6–8but not all investigators. 5,9Because the mechanisms of action of osmotic and loop diuretics differ, some have hypothesized that a combination of drugs might be uniquely beneficial. Consistent with this idea, the combination of an osmotic and loop diuretic has been reported to produce a more marked or longer-lasting reduction of ICP. 4,5,10
The mechanism by which diuretics affect ICP and brain water content is still the subject of some debate. For many years, the beneficial effect of these drugs has been proposed to result from loss of body water with a proportional loss of brain water. This is supported by early studies relating the volume of urine produced to reduction of ICP. 5,6,11,12However, although many studies have focused on diuretic-induced loss of body water, the role of diuresis per se is unclear. For example, osmotic diuretics produce more marked and longer-lasting reduction of increased ICP in nephrectomized animals than in animals with normal renal function, indicating that excretion of water is not necessary to reduce ICP. 13In other experiments, furosemide produced marked loss of body water or body weight without alteration of brain water, indicating that reduction of peripheral water content alone may not affect brain water. 14,15Diuretics may also affect ICP by mechanisms independent of diuresis. For example, furosemide can alter regulation of intracellular water content in brain cells, and mannitol may alter brain blood volume. 16,17Thus, there are a number of mechanisms by which diuretics may reduce ICP.
One effect of mannitol is to increase plasma osmolality, causing water to move from brain into blood along an osmotic gradient, reducing brain water content, brain bulk, and ICP. 18–20Furosemide alone does not acutely alter plasma osmolality, and consistent with this, it does not alter brain water content. 3–9,21–23However, although others have examined the effects of these drugs (alone and in combination) on ICP and plasma osmolality, 4,5,10no studies have compared their combined effects on brain water content. One prior study reported that furosemide augmented mannitol-induced increase in plasma osmolality 11. Thus, it is possible that furosemide and mannitol might interact via an osmotic mechanism to result in a greater reduction in water content than with mannitol alone.
We hypothesized that if the combination of mannitol with furosemide produced a greater increase in plasma osmolality, then the combination of mannitol and furosemide would produce a greater decrement in brain water. We first evaluated the dose-response of mannitol and furosemide alone on plasma osmolality and on the water content of normal rat brain and then studied the interaction of the two drugs.
Materials and Methods
The University of Iowa Animal Care and Use Committee approved all experiments. Eighty-two male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN); 322 ± 19 g (mean ± SD) were anesthetized with 4% halothane in 100% oxygen in a plastic box. When unresponsive, animals were removed from the box, 1% lidocaine was infiltrated subcutaneously into the anterior neck, and a tracheotomy was performed. The lungs of the animals were subsequently ventilated with an inspired gas mixture of 1% halothane in 40% O2/60% N2. Neuromuscular blocking drugs were not used. Femoral arterial and venous catheters (PE-50, Becton Dickinson, Sparks, MD) were inserted, again after subcutaneous infiltration of lidocaine. Mean arterial pressure was continuously measured from the femoral artery. Rectal temperature was maintained at 37°C with a heating pad. After preparation, baseline arterial pH, PO2, and PCO2were measured and ventilator settings were adjusted to achieve normocapnia. At this time, baseline plasma osmolality was also measured in duplicate by freezing point depression; values are expressed as milliosmoles per kilogram water (mOsm/kg; model 3MO microsmometer; Advanced Instruments, Needham Heights, MA).
The initial component of the study addressed the dose-related effects of mannitol alone or furosemide alone on plasma osmolality and brain water content. Pilot studies used doses of mannitol of less than 1 g/kg. However, these doses did not significantly alter either plasma osmolality or brain water (data not shown). Therefore, subsequent animals were randomly allocated to receive mannitol (25%) in doses of 1, 4, or 8 g/kg (n = 8, 8, and 10, respectively; one dose per animal). Additional rats received furosemide (10 mg/ml) in doses of 2, 4, or 8 mg/kg (n = 7, 4, and 10 respectively, one dose per animal). Drugs were infused over 5 min, except for the largest mannitol dose (8 g/kg), which was given over a 15-min period to avoid the hemodynamic instability seen with more rapid administration. One hour after the completion of drug administration, pH, arterial blood gases, and plasma osmolality were again measured. Animals were killed with an overdose of halothane and decapitated, and the brain was rapidly removed. The brain—consisting of the cortical structures, cerebellum, and brain stem as a single piece—was weighed, desiccated at 80°C for 96 h, and then reweighed. Brain water content was calculated by wet–dry weight difference. A separate control group of animals (n = 10) were treated in a similar fashion but did not receive either mannitol or furosemide.
The second component of the study examined the interaction of mannitol and furosemide and was conducted after completion of the single-drug experiments. Animal preparation was identical to that in the first study. Animals were randomly allocated to receive a combination of 8 mg/kg furosemide and (1) mannitol, 1 g/kg (n = 7); (2) mannitol, 4 g/kg (n = 8); or (3) mannitol, 8 g/kg (n = 10). Systemic parameters and brain water were measured as in the first study.
All results are expressed as mean ± SD. Values in the control group were compared at the two measurement points by repeated-measures ANOVA. Values in experimental groups were compared with control values by means of ANOVA, with a Dunnett post hoc test. Plasma osmolality and brain water content values were compared at each dose of mannitol between the mannitol and mannitol-with-furosemide groups by t tests. Regression analysis was used to examine the relation of plasma osmolality to brain water, and analysis of covariance was used to test for a difference in the slope of the regression lines for mannitol alone and mannitol with furosemide. A value of P < 0.05 was accepted as significant.
Systemic variables in the mannitol and furosemide groups before and 1 h after intervention are shown in table 1. Before intervention, mean arterial pressure was greater in the 8 mg/kg furosemide and 1 g/kg mannitol-with-furosemide groups than in the control group (P < 0.05). After intervention, mean arterial pressure was slightly greater in the group that received 8 g/kg mannitol and in all three mannitol-with-furosemide groups, relative to that in the control group (P < 0.05). After intervention, the partial pressure of oxygen was also greater in some groups relative to that in the control group (P < 0.05).
Plasma Osmolality and Brain Water
Baseline plasma osmolality averaged 301 ± 3 mOsm/kg in the control group and did not change with time (table 1). Baseline plasma osmolality was not different between any treated group and the control group (table 1). After treatment with furosemide, plasma osmolality was not different than in the control group (table 1). However, in groups treated with mannitol, there was a progressive, dose-related increase in plasma osmolality, which was statistically greater than in the control group for the groups that received 4 or 8 g/kg mannitol (P < 0.05;table 1and fig. 1). In groups treated with the combination of mannitol with furosemide, plasma osmolality significantly increased relative to the control in the groups receiving 4 and 8 g/kg mannitol with furosemide (P < 0.05;table 1and fig. 1). Plasma osmolality was also significantly greater in each of the mannitol-with-furosemide groups than with mannitol alone (P < 0.05;table 1and fig. 1).
Control and treatment brain water contents are shown in table 1. Treatment with furosemide did not affect brain water content at any dose (P > 0.05). Brain water content was lower than for controls in groups treated with 4 and 8 g/kg of mannitol alone (P < 0.05;table 1and fig. 2). In groups that received the combination of mannitol and furosemide, brain water content was significantly lower than for controls among recipients of 4 and 8 g/kg of mannitol (P < 0.05;table 1and fig. 2). Brain water was significantly less in groups that received the combination of 4 and 8 g/kg of mannitol with furosemide than with mannitol alone at these doses (P < 0.05;fig. 2).
A scattergram of postintervention osmolality and brain water content is shown in figure 3. Separate regression lines are shown for the mannitol and mannitol-with-furosemide groups. Brain water content correlated with plasma osmolality in both the mannitol-alone groups (r2= 0.869) and in the mannitol-with-furosemide groups (r2= 0.956). The slopes of the regression lines for the mannitol-alone and mannitol-with-furosemide groups were not statistically different (P = 0.05).
The new finding of this study is that in normal rat brain, the combination of mannitol with furosemide produced a greater reduction in brain water content than did mannitol alone. The most likely explanation for this observation is the greater increase in plasma osmolality that occurred when furosemide was combined with mannitol. This idea is supported by the high degree of correlation between plasma osmolality and brain water in both the mannitol-alone and mannitol-with-furosemide groups.
Consistent with findings in previous studies, mannitol alone produced a dose-related decrease in brain water content, which was linearly related to a mannitol-induced increase in plasma osmolality. 6,19,20,24,25The effects of osmotic agents on brain bulk and ICP have been known since the early 1900s. 26Whereas diuretics have been described to reduce ICP by a number of actions, the primary mechanism by which osmotic agents reduce brain water content is related to the establishment of an osmotic gradient across the blood–brain barrier, with a shift of water from brain into blood. The relative impermeability of the normal blood–brain barrier to most solutes (e.g. , urea, sodium, glycerol, and mannitol) allows substantial osmolar gradients to exist between blood and brain. Gradients for water movement can be calculated from the van't Hoff relation, and a 1-mOsm/kg difference will generate the equivalent of a 19–mm Hg hydrostatic driving force for water movement. 27
In our study, furosemide alone did not affect plasma osmolality or brain water content. This is consistent with the findings of most prior investigations, in which furosemide did not influence plasma osmolality 2,5,9,14or brain water content, 3,9,12,14,15,21–23although some investigators have reported that furosemide selectively reduces water content of edematous brain. 1,6,12Furosemide has been reported by many 1–4,8,22but not all investigators 5,9to reduce increased ICP. The mechanism by which furosemide reduces ICP is unclear; ICP can be influenced by mechanisms other than reduction of brain water content. Some investigators have reported that clinical doses of furosemide (1–2 mg/kg) reduce formation of cerebrospinal fluid. 28,29However, much larger doses (50 mg/kg) are required to consistently reduce cerebrospinal fluid formation, possibly by inhibition of carbonic anhydrase. 30–33The half-maximal concentration of furosemide for inhibition of cerebrospinal fluid formation is approximately 33 mg/kg. 33Other investigators report that smaller doses of furosemide, which reduce ICP, do not alter formation of cerebrospinal fluid. 32,34,35Overall, it appears that clinically used doses of furosemide are unlikely to affect ICP via change in CSF production. Furthermore, such doses of furosemide do not alter plasma osmolality or water content of normal brain. The precise mechanism by which furosemide affects ICP is unclear.
Although we found that furosemide alone did not affect plasma osmolality or brain water, we found that furosemide in combination with mannitol produced a larger increase in plasma osmolality than did mannitol alone. Given this augmented change in osmolality, we were not surprised to see a statistically greater reduction in brain water in animals treated with the combination of mannitol and furosemide. Previous findings have suggested that furosemide can enhance the effect of mannitol. Mannitol with furosemide can produce a greater increase in serum osmolality, 11which is more sustained 5and associated with a longer-lasting reduction of ICP 4,36than mannitol alone.
The mechanism by which furosemide affects mannitol-induced change in plasma osmolality is not clear. Previous investigation has shown that administration of furosemide with mannitol does not alter plasma mannitol kinetics, so the enhanced plasma osmolality cannot be accounted for by delayed excretion of mannitol. 36Furosemide increases both urinary water and sodium loss, and plasma sodium is less with combination therapy than with mannitol alone. 11Thus, the increase in osmolality cannot be accounted for by enhanced excretion of water over solute, resulting in an increase in plasma osmolality by an increase in plasma electrolytes. The overall interaction of water and solute kinetics that results in greater plasma osmolality with combination therapy is not yet understood.
Furosemide and mannitol could interact to produce greater reduction of brain water via regulation of cell volume. During an acute increase in osmolality, cells shrink because of a reduction of intracellular water. However, this volume decrease is transient; volume-regulatory mechanisms, including the influx of chloride (which increases intracellular osmolality), return cell volume to normal. Furosemide impairs the active volume-regulatory mechanisms in brain cells by preventing the cells from restoring intracellular volume. 17,37This could result in a greater reduction of intracellular water content and overall greater reduction of brain water content. Although it is possible that furosemide enhanced the effect of mannitol via such a mechanism, we cannot specifically comment on this because it is beyond the scope of the current study.
Limitations of the Current Study
In the current study, we used large doses of both mannitol and furosemide. We selected these doses for several reasons. The goal of this study was to evaluate the potential interaction of mannitol and furosemide on brain water rather than to establish clinically useful drug doses. Because the doses we selected were quite large, furosemide-induced enhancement of the effect of mannitol on brain water cannot be assumed to occur at clinically utilized doses. We selected mannitol doses that produced a wide range of change in brain water content 1 h after administration. Pilot studies showed that smaller, clinically used doses of mannitol (e.g. , 0.2 and 0.5 g/kg) resulted in no significant change in either osmolality or water content in normal rat brain. Regression analysis of data from the current and previous studies indicates that the slope of brain water content versus plasma osmolality is approximately 0.03% H2O per mOsm/kg when both parameters are measured 1 h after mannitol administration. 38,39To alter water content by 0.71% (the smallest change we detected as significant), a plasma osmolality change of 24 mOsm/kg was required. Regression analysis of plasma osmolality versus mannitol dose from the current data yields a slope of 6.5 mOsm/gm mannitol (osmolality = 301 + 6.5 × g/kg mannitol; r2= 0.982). Thus, to change osmolality 24 mOsm would require 3.7 g/kg of mannitol. Because our animals had normal renal function, the kinetics of mannitol and effect on brain water are more complex than this single-point analysis. However, because of the good correlation among mannitol dose, plasma osmolality, and brain water content, the analysis illustrates the need for large osmolar gradients to measurably reduce water content of normal brain. Large osmolar gradients may be required because of the water permeability of the normal blood–brain barrier, which is two- to three-log–fold less permeable to water than the peripheral circulation. 16,40
It is also possible that larger doses of mannitol were required in our study because of differences in mannitol kinetics in rats and humans. We were unable to find published rat mannitol kinetics, although kinetics have been measured in other animals and are similar to human mannitol kinetics. 41Regression analysis of published human mannitol doses versus plasma osmolality (measured 1 h after mannitol administration) results in a slope of 7 mOsm/gm mannitol, 11,42–45a value similar to the slope calculated from our data for rats. This suggests that mannitol kinetics are likely similar in humans and rats. The ability of clinically utilized, smaller doses of mannitol to decrease ICP may be related to removal of small amounts of brain water that are less than the limits of detection with wet–dry weight differences. It is also possible that smaller doses of mannitol may affect ICP by processes other than water removal and potentially by several processes that act in combination.
Our furosemide doses were selected for reasons similar to those used to select the mannitol doses. Doses of furosemide utilized in humans are typically 1 mg/kg or less. 2,11Because small doses of furosemide did not alter brain water or plasma osmolality, we selected larger doses to enhance our ability to detect any effect. Our goal was to insure that the lack of effect of furosemide was not the result of inadequate dose.
A second limitation of our study was our decision to assess water content 1 h after drug administration. We selected this time on the basis of clinical observations; the acute administration of both diuretics has been reported to affect ICP within minutes, and a maximal effect of ICP occurs within 1 h. 3,5,7,10It is possible that measurements made either earlier or later than 1 h might show a different result.
The third and most important limitation is that this work was performed in normal animals, rather than in animals with some form of brain injury or disease. We felt that this was a reasonable starting point in view of the variety of different pathologic models that could be selected, as well as the lack of basic information concerning the interaction of mannitol and furosemide. The effect of increased osmolality in reducing brain water is thought to be limited to areas of the brain with functional blood–brain barrier. Hence, we believed that it would be more likely to observe an effect in normal animals, where the blood–brain barrier is fully functional. Nevertheless, we realize that some investigators have reported that mannitol or furosemide reduces water content of injured brain but not that of normal brain. 6,19,46
In summary, we found that a large dose of furosemide could interact with mannitol to produce a greater increase in plasma osmolality and a greater decrease in brain water than mannitol alone. Change in brain water is only one aspect of how diuretics can reduce ICP; how furosemide and mannitol reduce ICP in the clinical setting is not yet understood.