Cellular Responses to Osmotic Perturbation

Magnetic resonance imaging (MRI) relaxation and diffusion properties are sensitive to the physiological state of cells and tissues. In particular, cerebral ischemia and stroke is associated with a reduced apparent diffusion coefficient (ADC) and increased transverse magnetization relaxation parameter (T2) in proton (1H) MRI making it useful for clinical diagnosis. Further, increases in sodium (23Na) signal intensity in 23Na MRI correlate with several neurodegenerative diseases as well as ischemia and stroke, which can be used to identify stroke lesions and predict stroke onset time. However, the contributing mechanisms underlying these changes, including possible increases in intracellular sodium and/or lengthened 23Na T2 relaxation, are not well understood. It has been hypothesized that alterations in cell regulatory mechanisms result in cell swelling, which disrupts tissue microstructure and ionic distributions. Osmotic perturbations have been used on single neurons and neural tissue models to mimic these volume changes. Evaluated with 1H MRI, hypotonic perturbations mimic tissue ischemia and result in an increase in 1H signal intensity, a decrease in 1H ADC, and an increase in 1H T2, while all trends are reversed for hypertonic perturbation. Single cell work has focused on the large L7 neuron from the abdominal ganglion in the sea hare Aplysia californica, which has been shown to have distinct nuclear and cytoplasmic compartments with differing relaxation and diffusion properties. Therefore, it would be useful to develop a tissue model comprised of large cells in which the intracellular contribution to the volume averaged signal could be determined. In this study, Aplysia abdominal ganglia were used due to their simple anatomy with a small collection of relatively large neurons up to 300 microns in diameter representing a simple neural tissue model system. The abdominal ganglia were dissected from the living animal, washed with isotonic artificial sea water (ASW; in mM: 460 NaCl, 10.4 KCl, 55 MgCl2, 11 CaCl2, 15 HEPES) and loaded into a 2.5-mm o.d. capillary containing isotonic, hypertonic, or hypotonic ASW. Hypertonic and hypotonic perturbations were introduced by changing the isotonic sodium chloride concentration (460 mM) to 545 mM and 345 mM, respectively. All MR imaging was performed at 11.75 T utilizing a homebuilt, double-tuned 1H/23Na solenoidal coil having a diameter of 3 mm. Imaging was performed in separate studies to quantify the proton T1, T2, T2*, and ADC immediately following dissection on viable ganglia as well as sodium T1 and T2* after the loss of cell viability. Changes in the MRI relaxation parameters (T1, T2 and T2*) as well as the apparent diffusion coefficient (ADC) in isolated ganglia were used to assess changing cellular environments under osmotic perturbation in the context of the influence changing ionic distributions, notably sodium, have on cell swelling in disease states. With tonicity changes, the ganglionic sac remains intact although alterations in the signal intensity (particularly with respect to sodium) are evident. Proton T1 did not change significantly with osmotic perturbation; however, 1H T2 and T2* decreased with increasing tonicity and 1H T2* provided the most robust measurement for identifying cellular changes due to osmotic perturbation. All relaxation parameters increased with cell death and loss of viability and no changes in 1H ADC were observed. The trends observed in 1H relaxation are possibly due to an increased intracellular volume fraction while the lack of a clear trend in ADC values may be due to significant volume averaging between the nuclear, cytoplasmic, and interstitial environments and should be modeled utilizing a multi-compartmental approach. Sodium measurements reveal a general increasing trend in relaxation with osmotic perturbation but no clear conclusions could be drawn. Changes in sodium relaxation are evident in compromised ganglia; however, linearly increasing trends in sodium signal intensity are seen in all osmotic conditions and may represent changes occurring with loss of cell viability, providing evidence that the direct evaluation of ions may prove more sensitivity in assessing pathological osmotic changes. Future work with perfused ganglia as well as voxel-selective spectroscopy and contrast agents to isolate the intracellular sodium signal could be used to answer questions regarding these observed trends in sodium signal intensity and relaxation.