Astroglial Volume Changes Induced by Acute Osmotic Stress in Brain Slices
Slices with GFP-expressing astrocytes can be imaged in real-time to observe structural changes induced by osmotic stress. Astrocytes included in this study were imaged at a slice depth >50 μm from the cut surface. The majority were deeper than 100 μm where neuropil preservation is optimal (Davies et al., 2007; Kirov et al., 1999). Recently using 2PLSM in slices, we reported preliminary findings of astroglial responses to acute osmotic challenge (Andrew et al., 2007). Here osmotic responses from a total of 56 GFP-expressing astrocytes in 29 slices from 8 animals were analyzed in detail. In all experiments, baseline images were acquired in control ACSF (291–293 mOsm) for 15–20 min.
Eight slices from 3 animals were subjected to hypo-osmotic ACSF and then to hyper-osmotic ACSF, using mannitol, followed by return to control ACSF in a subset of slices (Fig. 1). A steady shift in the focal plane during the first 3 min of osmotic challenge confirmed an overall change in the slice volume. Astrocyte somata superfused with −40 mOsm ACSF for 15 min significantly increased in area as derived from MIP images and then significantly decreased from control following 15 min in +40 mOsm ACSF. Area measurements returned to control following 15 min in normosmotic ACSF (Fig. 1A1–A4). Astroglial swelling and shrinking were also detected when corresponding MIP image stacks acquired in hypo- and hyper-osmotic ACSF were overlaid (Fig. 1A5–A6). This paradigm was repeated in a total of 6 slices. In addition, 2 slices were exposed to either −50/+50 or −60/+60 mOsm ACSF followed by control ACSF.
Figure 1. 2PLSM observation of astrocytes provides no indication of active volume regulation during acute osmotic challenge. A1–A4: 2PLSM sequence of GFP-expressing astrocytes from CA1 in hippocampal slice. Astrocytes (A1, control) swell during a 15 min superfusion with hypo-osmotic ACSF (A2), shrink during a 15-min treatment with hyper-osmotic ACSF (A3), and then return to baseline volume during 20 min in control ACSF (A4). Hypo-osmotic (A5; red) and hyper-osmotic (green) images are overlaid (A6) with arrows pointing to red areas illustrating volume differences under these conditions. B1–B3: 2PLSM sequence of an astrocyte labeled with astrocyte-specific dye SR101. The astrocyte (B1, control) swells under acutely overhydrated conditions (B2) and shrinks when dehydrated (B3). C1–C3: 2PLSM sequence of a GFP-expressing astrocyte and dendrite from CA1 in hippocampal slice. The astrocyte (arrow), seen in control in C1, shrinks under hyper-osmotic stress (C2) and recovers in normosmotic ACSF (C3). The dendrite (asterisk), however, is unchanged. D1: Quantification of astroglial soma area changes from control to hypo-osmotic to hyper-osmotic conditions and then during return to control ACSF. The number of astrocytes analyzed in 8 slices from 3 animals is indicated for each condition. No active volume regulation was detected. Values are shown as percent change from control. Asterisks indicate significant difference from control (*P < 0.001). D2: Summary of measurements in 7 slices of 4 animals shifting from control to hyper-osmotic to hypo-osmotic conditions and then returning to control ACSF (*P < 0.01). No active volume regulation was detected. Values are shown as percent change from control. Asterisks indicate significant difference from control (*P < 0.01).
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Compared with control ACSF, mean astroglial area increased significantly by 17.9 ± 2.8% (P < 0.001, Fig. 1D1) during 17.7 ± 3 min of overhydration. During 17.1 ± 2.8 min of dehydration, this value decreased to 11.7% ± 1.5% below control (P < 0.001, Fig. 1D1) and returned to within 2% ± 2.7% of control (P = 1.0, NS from Control) during 15.8 ± 3.6 min of superfusion with normosmotic ACSF (Fig. 1D1). During 15–20 min of osmotic challenge, images were collected in the first few minutes when volume regulation is purported to compensate for osmotic swelling or shrinking (not shown). There was no indication of either RVD during hypo-osmotic challenge or RVI during hyper-osmotic treatment, suggesting that astrocytes passively changed their volume during osmotic challenge without any apparent active volume regulation toward baseline.
To further verify these observations, the order of the osmotic challenges was reversed in 7 slices from 4 animals. Slices were exposed to hyper-osmotic ACSF (+40 or +80 mOsm) and then to hypo-osmotic ACSF (−40 mOsm) followed by control ACSF. Previously we demonstrated that neurons and dendrites steadfastly maintain their volume during acute osmotic stress (Andrew et al., 2007). Figure 1C1–C3 shows a dendrite and astrocyte in hippocampal slice from a hybrid mouse expressing GFP in neurons and astrocytes. While the dendritic volume remained stable, the astroglial soma shrank during 15 min in hyper-osmotic ACSF (Fig. 1C2) and recovered during 15 min in normosmotic ACSF (Fig. 1C3). Relative to control, the mean astroglial soma area significantly decreased by 10.8% ± 1.2% (P < 0.001, Fig. 1D2) during 17.2 ± 2.3 min of hyper-osmotic stress, then increased by 9.1% ± 2.4% over control during 17.9 ± 2 min of hypo-osmotic treatment (P < 0.01, Fig. 1D2). During re-exposure for 17.4 ± 1.7 min to control ACSF, this value returned to within 4.1% ± 3.3% of baseline (P = 0.19, NS from Control, Fig. 1D2). Again these astrocytes showed no apparent volume regulation during the first minutes of osmotic change in this reversed sequence of challenges.
These astroglial responses to osmotic stress were replicated in 3 slices from 2 animals containing astrocytes stained with the astrocyte-specific marker SR101 as shown in Fig. 1B1–B3. Compared with GFP-expressing astrocytes, SR101 lightly stains processes and capillary end-feet but somata are fluorescent enough to measure their MIP area. Relative to control, the mean SR101-labeled astroglia soma area significantly increased by 17.0% ± 4.4% (P < 0.01, n = 4 astrocytes) during 17.5 ± 1.7 min of exposure to hypo-osmotic ACSF (−40 mOsm) and then decreased by 26.9% ± 9.1% (P < 0.05, n = 4 astrocytes) during 15.3 ± 3.5 min of hyper-osmotic stress (+40 mOsm). Thus experiments with SR101 confirmed that structural changes observed in GFP-expressing astrocytes were associated with passive volume change, and that the presence of transgene was not altering their osmotic response.
It is possible that astrocytes swell or shrink in the initial 5 min of osmotic challenge and then volume regulate during the next 15 min. Such compensation would not have been detected by sampling at 15–20 min (Fig. 1). Therefore, images were sampled following 5 min of osmotic challenge in 14 slices from 7 animals (Fig. 2). In a subset of experiments, slices from 3 mice of the enhanced GFP strain were used. Overlaying a soma in control and experimental ACSF at 5 min (Figs. 2A1,B1) revealed cell body swelling in hypo-osmotic ACSF (Fig. 2A2) and shrinking in hyper-osmotic ACSF (Fig. 2B2). Most importantly, astroglial area increased by 12.8% ± 1.1% of control (P < 0.001, Fig. 2A3) during 4.9 ± 0.2 min of exposure to −40 mOsm ACSF and decreased by 10.0% ± 1.1% of control (P < 0.001, Fig. 2B3) during 4.9 ± 0.1 min of exposure to +40 mOsm ASCF.
Figure 2. Acute osmotic challenge results in a rapid and passive astroglial response. A1,B1: 2PLSM images of GFP-expressing astrocytes from CA1 region in hippocampal slice during 5 min of hypo-osmotic (A1) and hyper-osmotic (B1) stress. A2,B2: Overlays showing the merged control (red) and experimental (green) images, with arrows pointing at green areas representing swelling during exposure to hypo-osmotic ACSF (A2) or at red areas representing shrinking during exposure to hyper-osmotic ACSF (B2). A3: Summary from 12 astrocytes in 6 slices from 6 animals showing no detectable active RVD following soma swelling in response to 5 min of hypo-osmotic stress. Values are shown as percent of control. Asterisk indicates significant difference from control (*P < 0.001). B3: Summary from 17 astrocytes in 8 slices from 5 animals showing no detectable RVI following soma shrinking in response to 5 min of hyper-osmotic stress. Values are shown as percent of control. Asterisk indicates significant difference from control (*P < 0.001).
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As the number of intracellular GFP molecules does not change during osmotic challenge, the fluorescence should be inversely related to the intracellular water concentration (Crowe et al., 1995; Piston et al., 1999). Indeed hyper-osmotic stress resulted in 8.6% ± 3.8% (P < 0.001) increase in the soma GFP signal as compared with the control (n = 19 astrocytes from 9 slices). Hypo-osmotic challenge resulted in 21.1% ± 7.6% (P < 0.001) decrease in the soma GFP signal relative to the control (n = 18 astrocytes from 6 slices).
Thus the cohort of cells sampled at 5 min (Fig. 2) display volume increases and decreases similar to cohorts sampled at 15–20 min (Fig. 1). The absence of even a trend toward greater volume change measured at 5 versus 15–20 min indicates that volume changes are near-maximal by 5 min and that active volume regulation is not compensating during the intervening 10–15 min.
Astroglial Swelling Following Depolarization by Elevating [K+]O
We next used 2PLSM to determine if briefly elevating extracellular potassium would swell astrocytes, in conjunction with their role in the uptake of excess [K+]O. We exposed CA1 hippocampal astrocytes in 6 slices from 3 animals to 26 mM K+ ACSF for 3 min (Fig. 3). The image sequence of an astrocyte in control, 26 mM K+ ACSF and return to control ACSF (wash) is presented in Fig. 3A1–A3. Overlaying MIP images from control and elevated [K+]O conditions (Fig. 3A4) as well as during wash (Fig. 3A5) facilitated visual comparison between treatments. Astrocytes swelled as measured at 3.6 ± 0.4 min after the introduction of the 26 mM K+ ACSF by 38.9% ± 13% (P < 0.001, Fig. 3B) and then recovered following a 20 min wash (8.7% ± 9%, P = 0.35, NS from Control, Fig. 3B). We conclude that astrocytes swell reversibly in a manner consistent with their ability to buffer excess [K+]O.
Figure 3. Astrocytes swell during high-K+ treatment and recover in control ACSF. A1–A3: 2PLSM sequence of a GFP-expressing astrocyte from CA1 region. The astroglial soma and processes (A1, control) swell during 3 min of exposure to 26 mM K+ ACSF (A2) and return to control morphology after 20 min in control ACSF (A3). A4: Overlay showing the merged control (A1, red) and experimental (A2, green) images. Arrows point to green areas representing swelling during exposure to high K+ ACSF, which then reverses (A5). B: Summary from 12 astrocytes in 6 slices from 3 animals showing astroglial soma swelling induced by 3 min exposure to 26 mM K+ ACSF. Values are shown as percent of control measurements. Asterisks indicate significant difference from control (*P < 0.001).
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Astroglial Swelling and Partial Recovery Following Simulated Stroke in Slices
The reversibility of both osmotic and K+-evoked swelling of astrocytes generated confidence that glial responses to stroke-like events could be reliably imaged with 2PLSM. In particular, AD occurs within minutes of stroke onset, causing acute neuronal death (Kaminogo et al., 1998). To clearly delineate the morphological changes that occur within astrocytes following AD, we first used a brain slice model of OGD to simulate global ischemia (Obeidat and Andrew, 1998). Imaging of astrocytes and dendrites revealed swelling of both glial somata and dendritic processes following OGD (Fig. 4A). In addition, swollen dendrites lost their spines and became beaded (Fig. 4A2), a neuronal injury that immediately follows AD both in vitro (Andrew et al., 2007; Davies et al., 2007) and in vivo (Murphy et al., 2008).
Figure 4. Astrocytes and neurons swell and dendrites bead in response to oxygen/glucose deprivation. A1,A2: 2PLSM images of a GFP-expressing astrocyte (arrow) and dendrites (asterisk) from CA1 region before (A1) and after 10 min exposure to OGD, resulting in astroglial soma swelling and dendritic beading (A2). The region is cropped to show the same field before and after OGD. B1,B2: 2PLSM images of a GFP-expressing neuron (green) and astrocytes stained with SR101 (red) in a slice from somatosensory cortex. The neuronal (asterisk) and astroglial somata (arrows) seen in control (B1) become swollen and dendrites bead as the entire field expands during 10 min of OGD (B2). C1–C6: 2PLSM time sequence of a GFP-expressing astrocyte from CA1 during OGD. Time of OGD exposure is indicated within each image. The soma is swollen within 5 min (C4) and remains swollen for the entire 10 min exposure (C6). D1–D6: 2PLSM time sequence of an astrocyte and adjacent dendrite exposed to OGD (control in D1; subsequent images show time stamps during 10 min of OGD). The astrocyte swells around 4.5 min (D3) and the dendrite beads during the next minute (asterisks; D4–D6). E: Summary from 22 astrocytes in 10 slices from 6 animals showing the effect of 10 min of OGD on astroglial soma area. Values are shown as percent of control. Asterisk indicates significant difference from control (*P < 0.001). F: Graph of the events depicted in C showing the time-course of astroglial soma swelling during OGD. Values are shown as percent of control. Asterisk indicates significant difference from control (*P < 0.05, ** P < 0.01).
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Tissue swelling evoked by OGD for 10 min was accompanied by a progressive shift in the imaging focal plane starting at about 4 min. Ten slices from 6 animals showed a 29.0% ± 6% astroglial area increase from control measurements (P < 0.001, Fig. 4E). Similar increases in soma size of SR101-labeled astrocytes confirmed a volume increase not confined to GFP-expressing astrocytes (Fig. 4B). Neuronal cell bodies simultaneously swelled during OGD (Figs. 4B and 5B) as previously quantified (Andrew et al., 2007).
Figure 5. Astrocytes, but not neurons, show recovery from OGD-induced swelling in cortical slices. A1–A4: 2PLSM sequence of a GFP-expressing astrocyte from CA1 region of hippocampal slice. The astroglial soma (A1, control) swells during 10 min of OGD (A2). The swelling rapidly reverses during 1 min of exposure to control ACSF (A3), and volume remains constant during the next 15 min of re-oxygenation (A4). B1–B3: 2PLSM sequence of a GFP-expressing pyramidal neuron. The neuronal soma (B1, control), swells during 10 min of OGD (B2) but does not recover following 77 min of re-oxygenation (B3). C1: Summary from 12 astrocytes in 9 slices from 5 animals showing the effects of 10 min OGD and subsequent re-oxygenation/normoglycemia on astroglial soma area. Values are shown as percent of control. Asterisk indicates significant difference from control (*P < 0.01). C2: Summary from 7 neurons in 7 slices from 6 animals showing the effects of 10 min OGD and subsequent re-oxygenation (36.7 ± 26.5 min) on neuronal soma area. Values are shown as percent of control. Asterisks indicate significant difference from control (*P < 0.001).
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Having confirmed real-time astroglial swelling in response to OGD, we looked at its time-course in more detail. Using 9 slices from 5 animals, we acquired images of single optical sections of 12 GFP-expressing astrocytes to derive a time-course for soma swelling during 10 min of OGD (Fig. 4C). Images were taken once every minute for 10 min. Astrocytes started to swell at 5 min (Fig. 4C4) (10.7% ± 4%, P < 0.01, Fig. 4F) and remained swollen for the entire 10 min period (Figs. 4C6,F) (10.6% ± 3%; P < 0.05, Fig. 4F). Using a similar sequence taken from hybrid mice, we were able to compare the time-course of astroglial swelling with that of dendrites (Fig. 4D). The dendritic response to OGD followed the astroglial response closely (Fig. 4D4). These findings suggest that glial and neuronal swelling reliably occur in the first few minutes following OGD in vitro.
We next used 2PLSM to determine the extent of astroglial and neuronal recovery from ischemia. Ten minutes of OGD reliably induced cell swelling, so we introduced a post-OGD period of re-oxygenation/normoglycemia, using control ACSF, and recorded the changes in soma area during this recovery period (Fig. 5). Nine slices from 5 animals showed that astroglial soma area significantly increased during 10 min of OGD (20.0% ± 13%, P < 0.01, Figs. 5A2,C1) but rapidly returned to near baseline within 1 min of re-exposure to control ACSF (−0.5% ± 6%; P = 0.12, NS from Control, Figs. 5A3,C1). Seven slices from 6 animals showed neuronal soma swelling by 30.5% ± 19% during OGD (P < 0.001, Figs. 5B2,C2) but no recovery during re-oxygenation (35.5% ± 19%, P < 0.001, Figs. 5B3,C2). We conclude that although astrocytes and neurons swell similarly in response to global ischemia in slices, only astrocytes have the ability to recover morphologically to some degree.
Astroglial Swelling Observed In Vivo
After monitoring astroglial volume changes in slices, we then used noninvasive in vivo imaging to examine changes in native astrocytes concurrently with changes in blood flow. Cranial windows allowed visualization of blood vessels and GFP-expressing astrocytes within living mice. We first wanted to confirm that the osmotic stress-induced swelling seen in slices could be observed in vivo. Astrocytes were imaged in layer II/III of somatosensory cortex at a depth of about 100 μm (Figs. 6A,B). Hypo-osmotic stress was induced by IP distilled water injection (150 mL/kg). 2PLSM images obtained from 10 animals revealed rapid astroglial soma swelling during the initial 6 min following water injection (Fig. 6C). Soma area increased by 9.7% ± 10% (P < 0.05, Fig. 6C) at 3.0 ± 0.3 min and by 21.1% ± 13% (P < 0.001, Fig. 6C) at 9.5 ± 0.3 min (Figs. 6A2,B2). The increased soma size persisted throughout the 12–30 min period following water injection (P < 0.001, Fig. 6C), emphasizing a lack of RVD in vivo, confirming our slice findings.
Figure 6. Astrocytes swell in vivo following intraperitoneal water injection. A1,A2: 2PLSM images of an astrocyte (green) from layer II/III of somatosensory cortex with the soma surrounding a blood vessel (red) labeled with Texas Red dextran. The astrocyte (A1, control) is swollen at 10 min after IP water injection (A2). B1,B2: An astrocyte whose soma does not directly contact a blood vessel (but does make contact via end-feet, confirmed by following processes in z-series) is swollen by 8 min after water injection. C: Summary from 21 astrocytes from 10 animals showing increase in astroglial soma area following water injection. Values are shown as percent of control. Asterisk indicates significant difference from control (*P < 0.05, **P < 0.001).
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Out of the 21 astrocytes included in this analysis, only 4 fell into the category of “perivascular astrocytes;” i.e., an astrocyte whose soma directly contacted a blood vessel (Fig. 6A1). The remaining 17 astrocytes contacted blood vessels via processes/end-feet. During our imaging of layers II/III of somatosensory cortex, we observed that all astrocytes contacted at least one blood vessel via their soma or a process. The rapid soma swelling observed in this study was not restricted to perivascular astrocytes (Fig. 6A1,A2, and Supp. Info. Fig. 2), but also occurred in those with the soma distant from the blood vessel (Fig. 6B1,B2, and Supp. Info. Fig. 2).
As astroglial responses to osmotic stress in vivo nicely correlated with those in slices, we next examined if astrocytes would react to ischemic stress in the same manner. As shown in Fig. 4, AD induced by OGD resulted in astroglial soma swelling within 5 min in vitro. In our in vivo model, CA induced global ischemia, likely causing AD of neurons and glia within 1–3 min (although onset was not measured) (Chuquet et al., 2007; Murphy et al., 2008). Images obtained before CA (>50 μm deep in layer II/III of somatosensory cortex) were used as controls with subsequent images acquired at varying times after CA (Fig. 7). We were able to confirm CA by monitoring heart rate and visually by the loss of “streaking” in vessels (representing flow of non-fluorescent red blood cells) (Fig. 7A1,A2). In 6 animals imaged at 8.9 ± 0.6 min following CA, somata swelled similarly to the glial response to global ischemia in slices (Fig. 7A) (33.3% ± 8%, P < 0.01, Fig. 7B1). Astroglial processes also swelled (Fig. 7A), although these changes were not quantifiable because of the resolution limitations and brightness of GFP signal. However, the arbor that defines an astroglial domain (Bushong et al., 2002) did not change in size despite obvious swelling in the processes themselves (Fig. 7A) (3.2% ± 4%; P = 0.808, NS from Control, Fig. 7B2). Thus, astroglial swelling induced by global ischemia in vivo provided similar results to ischemia in slices, supporting our in vitro experiments.
Figure 7. Astrocytes swell in vivo following global ischemia induced with cardiac arrest by air embolization. A1,A2: 2PLSM images of an astrocyte (green) from layer II/III of somatosensory cortex and blood vessels (red). Blood flow within a capillary in control (A1, arrow) is indicated by streaking caused by scanning of moving non-fluorescent red blood cells. Blood flow stalls following cardiac arrest (A2, arrow) accompanied by swelling of astroglial soma and processes. Despite process swelling (A2), the area of the astroglial arbor remains constant. The blue outline (A1,A2) indicates the perimeter of the visible astroglial domain. B1: Summary showing an increased astroglial soma area after global ischemia. Values are percent of control from 14 astrocytes from 6 animals. Asterisk indicates significant difference from control (*P < 0.01). B2: Summary of measurements of the same astrocytes from B1, showing constant area of the astroglial arbor following global ischemia (P = 0.808). Values are shown as percent of control.
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