Dead-space microdomains hinder extracellular diffusion in rat neocortex during ischemia

Abstract

During ischemia, the transport of molecules in the extracellular space (ECS) is obstructed in comparison with healthy brain tissue, but the cause is unknown. Extracellular tortuosity (lambda), normally 1.6, increases to 1.9 in ischemic thick brain slices (1000 microm), but drops to 1.5 when 70,000 Mr dextran (dex70) is added to the tissue as a background macromolecule. We hypothesized that the ischemic increase in lambda arises from diffusion delays in newly formed dead-space microdomains of the ECS. Accordingly, lambda decreases when dead-space diffusion is eliminated by trapping dex70 in these microdomains. We tested our hypothesis by analyzing the diffusion of several molecules in neocortical slices. First we showed that diffusion of fluorescent dex70 in thick slices declined over time, indicating the entrapment of background macromolecules. Next, we measured diffusion of tetramethylammonium (TMA+) (74 Mr) to show that the reduction of lambda depended on the size of the background macromolecule. The synthetic polymer, 40,000 Mr polyvinylpyrrolidone, reduced lambda in thick slices, whereas 10,000 Mr dextran did not. The dex70 was also effective in normoxic slices (400 microm) after hypoosmotic stress altered the ECS to mimic ischemia. Finally, the dex70 effect was confirmed independently of TMA+ using fluorescent 3000 Mr dextran as a diffusion marker in thick slices: lambda decreased from 3.29 to 2.44. Taken together, these data support our hypothesis and offer a novel explanation for the origin of the large lambda observed in ischemic brain. A semiquantitative model of dead-space diffusion corroborates this new interpretation of lambda.

Figure 1.

Background macromolecules exclude dead-space diffusion: a hypothesis. Brain tissue is composed of cells surrounded by a thin layer of the ECS. Because the interstitial spaces are interconnected, they form a system of channels where signaling molecules and substances diffuse (top). During ischemia and other pathological conditions, cellular elements expand their volume as water moves from the extracellular to the cellular compartment, and blockages are formed in some interstitial planes. Diffusing molecules that enter these pocket-like regions are delayed and tortuosity increases (bottom left). When background macromolecules, such as dex70, are added to this tissue, they become trapped in the dead spaces. By excluding the dead-space volume, dex70 prevents marker molecules from being delayed there, and tortuosity decreases (bottom right). See the mathematical model in Materials and Methods for more detail.

Figure 2.

Trapping of dex70 in thick slices. The diffusion of fdex70 in a thick slice of neocortex was measured for 2 hr using the IOI method. Over time, an increasing number of macromolecules became immobile, which was detected as an apparent decrease in D^*. Three-dimensional representations of concentration of fdex70 shown in pseudocolor (red highest, blue lowest) taken immediately after the injection (0 min) and 24, 76, and 100 min later are shown above the graph. The time course of D^* was estimated by evaluating the diffusion process over 4 min intervals between any two successive image acquisitions (see Methods and Materials for details).

Figure 3.

Size of background macromolecules is critical for the decrease in λ in the thick slice. A, Representative examples of TMA⁺ diffusion curves recorded in neocortical thick slices incubated in ACSF containing pvp40 or dex10. A pulse of TMA⁺ (+60 nA iontophoretic current) was applied for 50 sec (horizontal bar), and its concentration was measured with a TMA⁺-ISM positioned 100 μm away. Recorded curves (solid line) are superimposed with the appropriate theoretical curves (dotted line). Measurements were done at 34°C at which temperature D for TMA⁺ is 1.24 × 10^-5 cm²/sec. The parameters of the ECS in the thick slice incubated with dex10 were λ = 1.88, α = 0.148, and k′ = 1.3 × 10^-6 sec^-1 for an iontophoretic microelectrode transport number n_t = 0.44. The parameters of the ECS in the thick slice incubated with pvp40 were λ = 1.63, α = 0.111, and k′ = 4.4 × 10^-3 sec^-1 with n_t = 0.48. B, C, Summary of tortuosity (B) and volume fraction (C) measured in the thick slices without background macromolecules (empty bars) and in the presence of dex10, pvp40, and dex70 (hatched and filled bars). The decrease in both parameters observed in the presence of pvp40 and dex70 was statistically significant (asterisks). [Data measured in the presence of dex70 are from Hrabětová and Nicholson (2000)].

Figure 4.

Background macromolecule dex70 reduces λ during hypoosmotic stress. A, Representative examples of TMA⁺ diffusion curves recorded in the neocortex of 400 μm slices. TMA⁺ pulse (+60 nA iontophoretic current) was applied for 50 sec (horizontal bar), and the concentration was measured with a TMA⁺-ISM positioned 120 μm away. Recorded curves (solid line) are superimposed with the appropriate theoretical curves (dotted line). The measurements were done at 34°C at which temperature the free diffusion coefficients are 1.24 × 10^-5 and 1.13 × 10^-5 cm²/sec in the absence and presence, respectively, of background macromolecule dex70. The iontophoretic transport number for all three records was n_t = 0.4. During hypoosmotic stress in dex70-free ACSF (no macro.), λ = 1.86, α = 0.139, and k′ = 2.3 × 10^-3 sec^-1. In the presence of dex70 (dex70), λ = 1.62,α = 0.112, and k′= 5.1 × 10^-3 sec^-1. The ECS parameters of the same slice before the challenge were λ = 1.65, α = 0.216, and k′ = 7.6 × 10^-3 sec^-1. B, C, Summary of tortuosity (B) and volume fraction (C) measured in 400 μm slices during hypoosmotic stress without background macromolecules (empty bars) and in their presence (hatched and filled bars). During hypoosmotic stress, both λ and α were significantly lower (asterisks) in the presence of dex70.

Figure 5.

Background macromolecule dex70 enhances the diffusion of fdex3 in the thick slices. A, Images (top) of fdex3 taken immediately after the pressure injection (labeled as 0 sec) and at 60, 120, and 180 sec later in neocortical thick slices at 34°C. The intensity shown in pseudocolor (red highest, blue lowest) represents the concentration of the fdex3 in the tissue. The images in the top row were taken in the absence of background macromolecules (no macro.). The images in the bottom row were in the presence of dex70 (dex70). The intensity profiles of data (bottom), obtained along the horizontal line running through the center of the image, are superimposed with theoretical curves (black dotted lines). In the presence of dex70, the image intensity dissipated faster, and therefore the collapse of the intensity curve (blue) is more pronounced. Tortuosities were 3.66 and 2.37 in the absence and presence of dex70, respectively. B, Summary of tortuosity measurements in the thick slices without background macromolecules (empty bar) and in the presence of dex70 (hatched bar). The tortuosity was significantly lower (asterisk) in the presence of dex70.