Changes in intracellular water diffusion and energetic metabolism in response to ischemia in perfused C6 rat glioma cells

Abstract

This work reports results of experiments in hollow-fiber bioreactor C6 glioma cell cultures where the apparent diffusion coefficient (ADC) of intracellular water (iADC) was measured at diffusion times between 0.83 and 40 ms. The experiments were carried out before and after the onset of permanent ischemia. The changes in iADC following ischemia were dependent on the diffusion time employed in the experiment. An ischemia-induced decrease in the iADC was measured at short diffusion times, while at long diffusion times the iADC increased. Decreases in the iADC measured at short diffusion times are interpreted to be a result of a decrease in the intrinsic diffusivity of intracellular water due to energy failure. Increases in iADC measured at long diffusion times, are interpreted to result from cell swelling.

Figure 1

A) Time-of-flight angiography through the center of the HFBR, showing a uniform delivery of media through the porous hollow fibers. B) DWMRI at b = 2000 s/mm², revealing the distribution of cells within the bioreactor.

Figure 2

PRESS pulse sequence with added PGSE or OGSE diffusion gradients. Diffusion gradients are applied simultaneously in both the Y and Z directions. In the PGSE diffusion waveform, Δ_eff is the duration between the gradients, and δ_eff is the duration of the gradient pulse. In the OGSE diffusion waveform, T is the length of the waveform, and N is the number of lobes, displayed here with N = 4.

Figure 3

¹H spectra from PRESS localized spectroscopy (TE = 16 ms, TR = 2500 ms), of the HFBR showing a splitting of the water signal into three resonances corresponding to water in the extracellular and intraluminal space (+200 Hz), water residing in the fiber wall (+135 Hz), and intracellular water (+15 Hz). Spectra are shown that were obtained pre-ischemia, and 20, 40 and 120 min post-ischemia.

Figure 4

Intracellular water signal vs. b-value at Δ_eff = 0.83, 5 and 30 ms. Solid black lines represent single exponential fits to the data.

Figure 5

A) Diffusivity of a water phantom at 25°C and iADC (pre-ischemia) vs. effective diffusion time. The diffusivity within the water phantom is used to calibrate iADCs to offset the effects of eddy currents. B) iADC at Δ_eff = 0.83 (OGSE), 10, and 30 ms (both PGSE) vs. time post ischemia. Pre-ischemia values are shown at 0 min +/− SD. The iADC at short Δ_eff decreases after ischemia, while it increases at long Δ_eff. At an intermediate diffusion time, Δ_eff = 10 ms, no significant change in the iADC is observed post-ischemia. C) Calibrated iADC vs effective diffusion time (pre- and 50 min post-ischemia). Data were fit to the Balinov et al. model of intracellular diffusion, and fits are shown as black lines.

Figure 6

D_int and β-NTP (A) and restriction length (B) parameterized from the Balinov et al. model plotted vs. time post ischemia. The estimated D_int decreases 32%, from 2.5 µm²/ms pre-ischemia to about 1.7 µm²/ms in the first 50 min post-ischemia. The fitted restriction length increases from 12 µm to about 14 µm post ischemia.

Figure 7

Intracellular and extracellular T₂ relaxation times from CPMG measurements vs. time post ischemia. Intracellular T₂ increases from 26 ms to 41 ms, while extracellular T₂ decreases from 36 ms to about 30 ms post-ischemia. At t = 0, the bioreactor is still flowing and the extracellular T₂ is not measureable.