Cerebral edema is one of the well recognized potentially serious complications of cerebral ischemia [¹]. While edema is believed to be an important factor of the high morbidity and mortality of large territory ischemic strokes, clinical options for treating cerebral edema remain limited [²]. Accumulated evidence now indicates that aquaporin 4 (AQP4), the most abundant water channel in brain, plays an essential role in the pathogenesis of cerebral edema [³]. AQP4 belongs to the aquaporin family of transmembrane water and water/glycerol transporters [⁴] and is found in high concentrations in mammalian astrocytes, particularly in the periventricular region and subpial endfeet [⁵]. Genetic deletion of AQP4 ameliorates brain swelling due to ischemia [⁶]. Transgenic mice lacking the dystrophin-associated protein complex, by which end-foot pooled AQP4 is anchored [⁷], show less severe ischemic edema compared to wild-type mice [⁸]. Genetic deletion of α-syntrophin, which is a member of the dystrophin-associated protein complex, also reduces ischemic edema [⁹, ¹⁰].

Recently, we identified several chemical structures with AQP4 inhibitory effect [¹¹, ¹²]. Using an in vitro bioassay, 2-(nicotinamide)-1,3,4-thiadiazole, TGN-020, was found to be the most potent AQP4 inhibitor among the agents studied [¹³]. In this study, we investigated the in vivo effects of TGN-020 on cerebral edema in a mouse model of focal cerebral ischemia using 7.0-T magnetic resonance imaging (MRI) in an attempt to find new pharmacological agents effective for treating ischemic cerebral edema.

Physiological parameters and rCBF following ischemia induction monitored by laser-Doppler flowmetry were virtually identical between control and TGN-020 treated groups (Table 1). Three out of 17 mice did not fit the inclusion criteria (one mouse exhibited respiratory distress with an SpO₂ under 94%, while the other two did not meet the minimum neurological deficit score). Thus, seven animals each of the vehicle and TGN-020 administered groups were analyzed by MRI. All mice meeting the inclusion criteria survived to the end of the MRI measurements.

Representative FSE images are shown in Fig. 1. Results are summarized in Fig. 2. The %BSV of the TGN-020 group (12.1 ± 6.3%) was significantly smaller than that of the control group (20.8 ± 5.9%). Likewise, the %HLV of cortex in the TGN-020 group (20.0 ± 7.6%) was also significantly smaller than that in the control group (30.0 ± 9.1%). The basal ganglia infarction volume in treated and control animals showed essentially no difference (Table 1). All sham operated mice showed no apparent infarction area.

The current study convincingly demonstrated that pretreatment with the AQP4 inhibitor TGN-020 significantly reduced the volume of brain edema associated with ischemic injury. Ischemic edema is believed to be initiated by influx of Na⁺ associated with energy failure. Higher osmolarity conditions create the driving force for water influx into cells, resulting in ionic edema [¹⁷]. This early edema phase, so-called cytotoxic edema, is believed to last several hours before bulk leakage of water into the brain ensues, producing so-called vasogenic edema [¹⁸]. AQP4 is believed to play a significant role in the actual water flux in both processes.

Flux through AQP4 is bidirectional and purely dependent on osmolarity differences between the two spaces connected by AQP4. Theoretically, pretreatment with an AQP4 inhibitor can block water movement through these water channels, thereby reducing the main water influx associated with influx of Na⁺, and delaying the development of full-fledged ionic edema. Although speculative, halting the early process mediating brain edema, may interrupt the subsequent cascade of events that lead to further edema such as astrocytic process swelling, up-regulation of astrocytic volume-regulated anion channel (VRAC) [¹⁹, ²⁰], or further influx of osmotically active ions [¹⁸]. An interesting observation in this study was the differential effect on infarct size by AQP4 inhibition. Whereas TGN-020 significantly reduced the cortical infarct volume (p < 0.05), it essentially had no effect on basal ganglia infarct volume. While entirely conjectural, the pathophysiologic mechanism underlying the observations may reflect the differential distribution of AQP4 channels in perivascular regions and cortical surfaces [³]. It is plausible that the difference in infarction volume between control and AQP4 treated animals denoted the effect of edema on infarction, and, therefore, represented potentially salvageable tissue by AQP4 inhibition. On the other hand, the similar %HLV, reflecting infarct volume for basal ganglia in both control and treated animals as well as for cortex in treated animals only, likely represented the direct result of ischemia.

In conclusion, we assessed the in vivo effect of a novel AQP4 inhibitor, TGN-020. Pretreatment with TGN-020 significantly reduced the severity of brain edema associated with brain ischemia. Further studies that better simulate clinical treatment conditions are essential. Nevertheless, AQP4 inhibition is a novel approach in reducing cerebral edema, and appears promising for the development of a unique class of pharmacologic agents effective in the clinical treatment of brain ischemia.

The study was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology, and University of Niigata.

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