The water channel AQP4 is enriched in perivascular astrocyte membranes where it is densely packed in distinctive rafts called orthogonal arrays of proteins (24
). Judged by its peculiar mode of expression AQP4 is likely to be one of the most abundant molecules at the brain-blood interface and has been proposed to play a major role in edema associated with a variety of brain injury paradigms (11
). The functional role of the various pools of AQP4 is presently under investigation. By drawing advantage of a tailor made transgenic model, the present study provides new insight in the physiological and pathophysiological roles of perivascular AQP4. First, we show that this AQP4 pool mediates the anti-edema effect of osmotherapy with HS in a well-characterized model of ischemic stroke. Second, HS attenuates BBB disruption depending on the integrity of the perivascular AQP4 pool. Third, deletion of the perivascular pool of AQP4 alleviates post-ischemic tissue damage, with or without the administration of osmotherapy. These data provide important insights into the mechanisms underlying the beneficial effect of osmotherapy following ischemic stroke and support the idea that the perivascular pool of AQP4 might be a promising target for therapy aiming at ameliorating post-ischemic edema.
Although HS solutions were first investigated experimentally over 85 years ago (30
) and subsequently utilized clinically for small volume resuscitation in patients with shock (31
), of recent these solutions have received renewed attention as hyperosmolar agents and are being increasingly utilized clinically to alleviate cerebral edema in a variety of brain injury paradigms (2
). Because sodium chloride (reflection coefficient = 1.0) (2
) is completely excluded from brain regions with an intact BBB, it has been proposed that HS may be a more favorable osmotic agent than the conventional osmotic agent mannitol (reflection coefficient = 0.9) which has been the mainstay in clinical practice since 1960. We have previously demonstrated that induction of a hyperosmolar state with HS effectively attenuates ischemia-evoked cerebral edema without accentuating ischemia-evoked BBB breakdown (6
). In addition to other beneficial non-osmotic properties of HS solutions (5
), its anti-inflammatory action leading to less BBB breakdown has also been proposed to afford benefit (31
). Furthermore, HS may be a more desirable agent for maintaining a “euvolemic hyperosmolar” state in a variety of brain injury paradigms as opposed to the conventional osmotic agent, mannitol (5
In the present study, we used HS as a tool and explored if its osmotic action depends on the integrity of the perivascular pool of AQP4. Our hypothesis was that the latter AQP4 pool mediates the egress of water that is induced when the brain is exposed to HS. In preliminary experiments, we determined that brain edema is maximal at 48 hr in our model of transient focal ischemia (data not shown). As in our previous studies (6
), therapy with HS was used as a continuous infusion (1.5 mL/kg/hr) to maintain a euvolemic hyperosmolar state and to maintain a constant osmotic gradient sufficient to cause egress of water from the injured and non injured brain. This rate of infusion is commensurate with maintenance fluid requirement in the human (9
). Furthermore, we used HS as chloride: acetate mixture to avoid hyperchloremic acidosis that occurs with use of concentrated chloride solutions alone. As in our previous studies (6
), wet-to dry ratios comparisons were used as a simple and reproducible assessment of brain water in both ischemic and nonischemic hemispheres. A major conclusion of this study is that HS makes a difference (compared with NS) only in those situations where perivascular AQP4 is available for water egress. When perivascular AQP4 is lacking—because of postischemic down-regulation (32
) or following α-syn deletion—the beneficial effect of HS vs. NS is negated. The findings are thus consistent with our hypothesis that perivascular AQP4 mediates the egress of water induced by exposure to HS.
We observed less brain water content in naïve α-syn−/− mice as compared with their WT counterparts. The explanation for this dataset remains unclear at present as parallel experiments failed to reveal differences in the baseline level of serum osmolality.
There is a distinct possibility that sustained hypertonicity upregulates a variety of proteins including AQP4. We have recently demonstrated that total AQP4 proteinexpression is upregulated in the ischemic hemisphere in animals treated with HS as compared with normal salinetreated animals in a well-characterized rat model of permanent focal ischemia (8
). α-Syn deletion also caused a modest reduction in perivascular Kir4.1 in our previous study (16
). However, other molecules engaged in transport processes across the blood-brain interface (e.g., monocarboxylate transporter 1, glucose transporter 1, excitatory amino acid transporter 2, and the NaKCl2
cotransporter), were not affected in this study.
The mechanisms of cerebral edema following focal ischemia are complex and not completely elucidated. Historically, postischemic edema has been divided into a cytotoxic component secondary to energy failure and a delayed vasogenic component secondary to BBB breakdown with leakage of plasma constituents (33
). Other secondary mechanisms that have been shown to play a significant role in accentuating ischemia-evoked cerebral edema include impedance of cerebral venous return from cerebral swelling, intrahemispheric diaschisis (34
), inflammation accentuating BBB disruption (35
), neurohormonal responses (37
), and induction of growth factors (38
). Of recent, AQP4—the most abundant of the aquaporin water channels in brain—has been implicated in the pathogenesis of cerebral edema in a variety of brain injury paradigms including ischemic stroke (11
). AQP4 has been shown to facilitate resorption of excess fluid in vasogenic cerebral edema associated with brain tumor and contusive injury (20
) and bacterial abscess (19
). However, the specific role and function of the perivascular AQP4 pool remains to be fully elucidated. Analyses of gene-targeted animals that lack perivascular AQP4 have indicated that this AQP4 pool is rate limiting for the rapid water exchange that occurs between the blood and brain in the accumulation and resolution phases of brain edema (14
). Specifically, a selective deletion of the perivascular AQP4 pool by targeted disruption of_α-syn causes a pronounced decrease in the extent of brain edema after a transient ischemic insult (14
). α-Syn contributes to the organization of the dystrophin complex in astrocytes (39
) and is essential for anchoring of AQP4 to the perivascular endfoot membranes (24
In keeping with our previous studies (6
), regional brain water content was attenuated by 1% to 2% in both the ischemic and nonischemic hemispheres with HS treatment. This magnitude of reduction in water content translates to >90 mL reduction in human brain volume (a geometric function of water content) (4
). The translational significance of these findings is important, as reduction in water content of this magnitude can be life-saving for patients with poor intracranial elastance resulting from large hemispheric strokes. As noted above, no such effect was presently observed in α-syn−/−
mice, indicating that the egress of water from the brain is mediated by the perivascular pool of AQP4. This finding leaves us with a new concept as to the mechanism of osmotherapy: the osmotic agents act by setting up an osmotic gradient that drives water through a rate limiting pool of perivascular AQP4. This concept is consistent with the bidirectionality of water flux through AQP4 (15
). Using in vivo
multiphoton imaging we have recently demonstrated (41
) that hyponatremic edema causes a volume increase of astrocytes that are in close proximity to brain microvessels. These data confirm that astrocytes are sites of water entry and underline the importance of cellular (cytotoxic) edema in the buildup phase of brain edema. Our in vivo
imaging data thus support the concept that the perivascular AQP4 pool mediates exchange of water across the brain-blood interface.
AQP4 is also expressed in luminal and abluminal endothelial cell membranes, albeit in low amounts compared with the expression level in perivascular endfeet (18
). The endothelial pool of AQP4 is unchanged after targeted disruption of the α-syn−/−
). For water to enter the astrocyte compartment in the brain it has to pass from the capillary lumen through three plasma membranes (luminal endothelial, abluminal endothelial, and luminal perivascular) of which the latter normally contains the highest density of AQP4 water channels. However, when reduced by >90% following α-syn deletion (18
) the perivascular AQP4 pool seems to become rate limiting for water exchange—in either direction—at the blood brain interface. Passage of water in the narrow cleft between the astrocyte endfeet may be impeded in the α-syn−/−
mice because of the slight swelling of these endfeet (15
It needs to be emphasized that the water flux across the brain-blood interface is not mediated exclusively by AQP4. In addition to a slow diffusion through the plasma membrane, water is subjected to co-transport with ions and organic molecules. Such co-transport may occur, e.g., through monocarboxylate, glucose, and potassium/chloride transporters. We have examined the known water transporting molecules at the brain-blood interface and none of these (except perivascular AQP4) seems to be affected by disruption of the α-syn gene (18
). Thus, our data strongly suggest that the effects presently observed after α-syn deletion can be attributed to the loss of perivascular AQP4.
Our study has limitations. Rectal temperature was maintained at 37°C ± 0.5°C in all animals during surgical procedures by placing them on a warming blanket. Although we did not measure brain temperature in our experiments, previous work in our laboratory and by others have demonstrated that maintenance of body temperature at 37°C ± 0.5°C achieves physiologic levels of brain temperature. We did not measure and track serial serum sodium levels in our experimental paradigm. Although the murine model has several advantages because of inclusion of transgenic strain, it has a small blood volume that precludes these measures. Therefore, we opted to determine serum osmolality rather than serum sodium at the end of the experiment (48 hr postischemia). It is well known that tight glycemic control is critical for stroke outcome. Although our data demonstrates a wide variation in serum glucose, it is to be noted that there were no statistical differences in any of these values (48 hr postischemia). Furthermore, our data does not support that osmotherapy had any effect on serum glucose levels. We used young adult male animals in our study. On the average α-syn−/− mice were 3 months older than their WT counterparts in our study to ensure that animals were weight-matched. Body weight is the critical determinant of consistency of injury volume in our model of ischemic stroke because vascular anatomy varies with body weight. Our study cannot comment on the effect of age on the functional role of various domains of AQP4 following cerebral ischemia.
In the present study we did not observe robust improvements as in our previous studies in the rat (6
) in functional neurologic deficits with HS, although mortality was attenuated as compared with treatment with NS. Species differences and large injury volume with 90-min MCAO may account for the lack of robust differences in functional outcome. Nevertheless, the primary goal of this study was not to demonstrate efficacy of HS as in our previous studies, but to use it as a tool in understanding the mechanism of efflux of water from brain during osmotherapy. Our conclusions are also tempered by the possibility that the α-syn deletion could have affected the integrity of the BBB and that this could explain why the effect of HS was abrogated in the α-syn−/−
animals. Our data suggest that the BBB remains intact following α-syn deletion, in keeping with our previous studies (17
). However, a more detailed evaluation of BBB characteristics following α-syn deletion is warranted. Interestingly, after MCAO, treatment with HS significantly restored BBB function in WT mice but had no significant effect in α-syn−/−
mice. Infarct volume was significantly attenuated in α-syn−/−
mice compared with WT as demonstrated previously (15
). The explanation for this observation remains elusive at present, but it is likely that it reflects an alleviation of the secondary effects of edema.