Effect of Mannitol and Furosemide on Plasma Osmolality and Brain Water

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).

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.

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.