Mechanical strain injury increases intracellular sodium and reverses Na+/Ca2+ exchange in cortical astrocytes

Abstract

Traditionally, astrocytes have been considered less susceptible to injury than neurons. Yet, we have recently shown that astrocyte death precedes neuronal death in a rat model of traumatic brain injury (TBI) (Zhao et al.: Glia 44:140-152, 2003). A main mechanism hypothesized to contribute to cellular injury and death after TBI is elevated intracellular calcium ([Ca2+]i). Since calcium regulation is also influenced by regulation of intracellular sodium ([Na+]i), we used an in vitro model of strain-induced traumatic injury and live-cell fluorescent digital imaging to investigate alterations in [Na+]i in cortical astrocytes after injury. Changes in [Na+]i, or [Ca2+]i were monitored after mechanical injury or L-glutamate exposure by ratiometric imaging of sodium-binding benzofuran isophthalate (SBFI-AM), or Fura-2-AM, respectively. Mechanical strain injury or exogenous glutamate application produced increases in [Na+]i that were dependent on the severity of injury or concentration. Injury-induced increases in [Na+]i were significantly reduced, but not completely eliminated, by inhibition of glutamate uptake by DL-threo-beta-benzyloxyaspartate (TBOA). Blockade of sodium-dependent calcium influx through the sodium-calcium exchanger with 2-[2-[4-(4-Nitrobenzyloxy)phenyl]ethyl]isothiourea mesylate (KB-R7943) reduced [Ca2+]i after injury. KB-R7943 also reduced astrocyte death after injury. These findings suggest that in astrocytes subjected to mechanical injury or glutamate excitotoxicity, increases in intracellular Na+ may be a critical component in the injury cascade and a therapeutic target for reduction of lasting deficits after traumatic brain injury.

Fig. 1

Ratiometric live-cell imaging of SBFI-AM and Fura-2-AM. A–C: Ratiometric live cell fluorescent imaging of SFBI-AM in cultured astrocytes. A: 340/380 ratio image before mechanical strain injury, B: Pseudo-colored imaged with white outline cell (region of interest) indicating intracellular Na⁺ concentration before and after (C) mild (5.5-mm) mechanical strain injury. D: Representative tracings from mild, moderate, and severely strain injured astrocytes where intracellular calcium concentration ([Ca²⁺]_i) values were obtained via ratiometric live cell fluorescent imaging of Fura-2-AM. Note that the dotted line indicates 60-s lapse in recording where cells were removed from microscope to be injured. Imaging resumes 30 s after mechanical stretch injury. E,F: Pseudo-colored image with white outlined cell (region of interest) indicating [Ca²⁺]_i before and after (F) moderate (6.5-mm) mechanical strain injury.

Fig. 2

Effect of injury or L-glutamate on [Na⁺]_i. Intracellular sodium ([Na⁺]_i) was measured with ratiometric live cell fluorescent imaging of SBFI-AM in cultured astrocytes. A: Effect of mild (5.5-mm), moderate (6.5-mm), or severe (7.5-mm) mechanical strain injury on [Na⁺]_i. Pre-injury baseline [Na⁺]_i was 13.7 ± 1.6 mM. B: Effect of 5-min application of L-glutamate (10, 100, or 1,000 μM) on [Na⁺]_i. Base-line [Na⁺]_i was 11.7 ± 1.8 mM. *, [Na⁺]_i significantly increased over baseline value (time 0) at time-point specified; +, [Na⁺]_i significantly increased over baseline at all time-points measured (n = 3 separate experiments).

Fig. 3

Effect of mechanical strain injury or L-glutamate on cell death. Either mechanical strain injury (A) or L-glutamate application (B) was administered to cultured astrocytes. Cell death was indicated by propidium iodide (PrI) uptake expressed as percentage of total PrI uptake. *, PrI was significantly increased over control (uninjured or untreated) values at time-point specified (n = 3 separate experiments).

Fig. 4

Effect of TBOA on [Na⁺]_i and cell death after mechanical injury. Sodium-dependent glutamate uptake was inhibited with 100 nM TBOA before strain injury. [Na⁺]_i was measured with ratio-metric live cell fluorescent imaging of SBFI-AM in cultured astrocytes. Pre-injury application of TBOA reduced injury-induced increases in [Na⁺]_i after mild (5.5 mm; A), moderate (6.5 mm; B) or severe (7.5 mm; C) injury. Cell death (D) was indicated by PrI uptake and expressed as percentage of total PrI uptake after lysing cells with Triton X. TBOA reduced cell death in the severe (7.5-mm) strain injury group only (*). A–C: *, TBOA application significantly reduced [Na⁺]_i versus injury at time-point specified; +, [Na⁺]_i significantly reduced over injury at all time-points measured (n = 3 separate experiments). Data in non-TBOA conditions are from Fig. 1.

Fig. 5

Effect of TBOA on [Na⁺]_i and cell death after L-glutamate. Sodium-dependent glutamate uptake was inhibited with 100 nM TBOA before and during L-glutamate application. [Na⁺]_i was measured with ratiometric live cell fluorescent imaging of SBFI-AM in cultured astrocytes. Pre-glutamate application of TBOA reduced glutamate-induced increases in [Na⁺]_i after 100 μM (A) and 1 mM (B). TBOA significantly reduced cell death in the 1 mM condition (C). A,B: *, TBOA application significantly reduced [Na⁺]_i versus glutamate level at time-point specified; +, [Na⁺]_i significantly reduced over glutamate level at all time-points measured (n = 3 separate experiments). Data in non-TBOA conditions are from Fig. 1.

Fig. 6

Effect of mechanical strain injury on [Ca²⁺]_i and Na⁺/Ca²⁺ exchange. Intracellular calcium ([Ca²⁺]_i) was measured with ratiometric live cell fluorescent imaging of Fura-2-AM in cultured astrocytes. The sodium-calcium exchanger (NCX) was inhibited in the reverse direction with 100 nM KBR-7943 before strain injury. Pre-injury application of KB-R7943 did not change [Ca²⁺]_i after mild injury (5.5 mm; A), but modestly reduced [Ca²⁺]_i after moderate strain injury (6.5 mm; B), and more robustly reduced injury-induced [Ca²⁺]_i after severe injury (7.5 mm; C). Pre-administration of KB-R7943 also reduced propidium iodide (PrI) uptake in moderate or severely injured astrocytes measured 24 h post-injury (D, *). A–C: *, KB-R7943 application significantly reduced [Ca²⁺]_i versus injury level at time-point specified; +, [Ca²⁺]_i significantly reduced over injury level at all time-points measured (n = 3 separate experiments).

Fig. 7

Effect of L-glutamate on [Ca²⁺]_i and Na⁺/Ca²⁺ exchange. Intracellular calcium ([Ca²⁺]_i) was measured with ratiometric live cell fluorescent imaging of Fura-2-AM in cultured astrocytes. The sodium-calcium exchanger (NCX) was inhibited in the reverse direction with 100 nM KB-R7943 before L-glutamate application. Pre-glutamate application of KB-R7943 did not reduce [Ca²⁺]_i in 100 μM condition. After 1 mM L-glutamate application, KB-R7943 reduced [Ca²⁺]_i at some time-points (*). Pre-administration of KB-R7943 did not significantly reduce propidium iodide (PrI) uptake in either condition, although there was a nonsignificant trend (P = 0.052) toward reduction after 1 mM glutamate application. (n = 3 separate experiments).