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. 2011 Oct 19:8:143.
doi: 10.1186/1742-2094-8-143.

Differential aquaporin 4 expression during edema build-up and resolution phases of brain inflammation

Thomas Tourdias  1 , Nobuyuki Mori, Iulus Dragonu, Nadège Cassagno, Claudine Boiziau, Justine Aussudre, Bruno Brochet, Chrit Moonen, Klaus G Petry, Vincent Dousset
Affiliations

Differential aquaporin 4 expression during edema build-up and resolution phases of brain inflammation

Thomas Tourdias et al. J Neuroinflammation. .

Abstract

Background: Vasogenic edema dynamically accumulates in many brain disorders associated with brain inflammation, with the critical step of edema exacerbation feared in patient care. Water entrance through blood-brain barrier (BBB) opening is thought to have a role in edema formation. Nevertheless, the mechanisms of edema resolution remain poorly understood. Because the water channel aquaporin 4 (AQP4) provides an important route for vasogenic edema resolution, we studied the time course of AQP4 expression to better understand its potential effect in countering the exacerbation of vasogenic edema.

Methods: Focal inflammation was induced in the rat brain by a lysolecithin injection and was evaluated at 1, 3, 7, 14 and 20 days using a combination of in vivo MRI with apparent diffusion coefficient (ADC) measurements used as a marker of water content, and molecular and histological approaches for the quantification of AQP4 expression. Markers of active inflammation (macrophages, BBB permeability, and interleukin-1β) and markers of scarring (gliosis) were also quantified.

Results: This animal model of brain inflammation demonstrated two phases of edema development: an initial edema build-up phase during active inflammation that peaked after 3 days (ADC increase) was followed by an edema resolution phase that lasted from 7 to 20 days post injection (ADC decrease) and was accompanied by glial scar formation. A moderate upregulation in AQP4 was observed during the build-up phase, but a much stronger transcriptional and translational level of AQP4 expression was observed during the secondary edema resolution phase.

Conclusions: We conclude that a time lag in AQP4 expression occurs such that the more significant upregulation was achieved only after a delay period. This change in AQP4 expression appears to act as an important determinant in the exacerbation of edema, considering that AQP4 expression is insufficient to counter the water influx during the build-up phase, while the second more pronounced but delayed upregulation is involved in the resolution phase. A better pathophysiological understanding of edema exacerbation, which is observed in many clinical situations, is crucial in pursuing new therapeutic strategies.

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Figures

Figure 1
Figure 1
Time course of LPC-induced edema as assessed by ADC measurements. (A) Quantification of ADC values (median, Q1-Q3) revealed a biphasic evolution (ANOVA) with a first phase characterized by a rapid increase in water content (§, p = 0.006, Wilcoxon test) peaking at 3 dpi, corresponding to the active phase of inflammation. The second phase was characterized by water resolution (*, p = 0.015, ANOVA), with ADC values that returned to baseline during the formation of a glial scar. ADC values of sham rats were stable over time and were not different from those measured in the contralateral side of LPC rats. The dotted line is the median value over the 5 time points for the sham group. (B) Representative illustration of the time course with T2WI (left panel) and merged T2/ADC maps (right panel) of the same animal taken at three different time points (3, 7 and 14 dpi) with corresponding histology at 14 dpi (Luxol Fast Blue coloration). A large area of edema with high ADC values was seen at 3 dpi along the right internal capsule (arrow) and spread through the extramedullary lamina and medial lemniscus tracts toward the midline (arrowheads). The majority of the edema was resolved by 7 and 14 dpi, with a slight cavitation at the site of injection (*) with cerebrospinal-fluid-like ADC values. Histological evaluation of the lesion at 14dpi confirmed the small cavitation (*) and showed large demyelination of the white matter tracts in which edema was initially observed. The myelin fibers of the internal capsule, stained in blue, were outlined (dotted lines) and a loss of myelin was seen in the internal capsule and also in the other white matter tracts (arrowheads).
Figure 2
Figure 2
Edema build-up and resolution phase characteristics. (A) Representative samples of Evans blue extravasation from rats sacrificed at 1, 3, 7, 14 and 20 dpi. Widespread leakage at 1 dpi (arrow) progressively decreased with a restriction to the lesion site (3 and 7 dpi, arrows) followed by a complete restoration of the BBB integrity at the later time points (14 and 20 dpi). (B) and (C) are representative illustrations of MRI and histological features for rats explored at 1 dpi (B) and 20 dpi (C). During the edema formation phase (1 dpi, B), the T2 signal increased along the internal capsule up to the midline with high ADC values (similar pattern as in Figure 1, day 3). The corresponding histology showed important BBB permeability (IgG) and massive infiltration of ED1 + cells around vessels (**) in MRI-defined edematous areas (dotted lines) while astrocytes were faintly stained (GFAP). During the edema resolution phase (20 dpi, C), T2 and ADC signals were mostly normalized, with the only persistence of a small cavitation at the site of injection due to necrosis (*, similar pattern as in Figure 1, day 14). The corresponding histology showed a large area with hypertrophic and entangled astrocytes i.e., gliosis (GFAP) around the point of injection (dotted lines) while BBB leakage (IgG) had mostly resolved with much lower presence of ED1+ cells.
Figure 3
Figure 3
Quantitative features of edema build-up and resolution phases. Markers of BBB permeability (immunostaining of endogenous IgG extravasation and Evans Blue leakage) and pro-inflammatory cytokine (IL1β mRNA quantification) were found as early as 1 dpi (§, p < 0.05, Wilcoxon test) and were significantly increased during the build-up phase of the model compared to the resolution phase (*, p < 0.001, Mann Whitney). The resolution phase (7 to 20 dpi) was characterized by the formation of a glial scar with a significant increase of GFAP (mRNA quantification *, p < 0.05, Mann Whitney).
Figure 4
Figure 4
Inflammatory cell subtypes. Double labeling of ED1 (Alexa 488, green) and Iba1 (CY3, red) in the contralateral brain (A) and at the lesion site at 1 dpi (B) and 14 dpi (C). On the contralateral side (A), only resting microglia were stained with ramified thin processes and weak Iba1 immunoreactivity. During the edema formation phase (1 dpi, B), many round cells with both ED1 and Iba1 immunopositivity (arrows) were found around vessels (**) and were thought to be infiltrating macrophages, while some could also represent amoeboid microglia with a fully activated profile. At the periphery of the lesion, some activated microglia Iba + but ED1 - could also be observed (arrowheads). During the edema resolution phase (14 dpi, C), most cells were Iba1 + but ED1 - and showed highly branched processes corresponding to activated microglia.
Figure 5
Figure 5
Time course of AQP4 expression during edema formation and resolution. (A) Histological evaluation depicted an initial upregulation of AQP4 as early as 1 dpi (§, p = 0.003, Wilcoxon test) that plateaued at 1 and 3 dpi. A significant increase in AQP4 expression was found during the MRI-defined edema resolution compared to the MRI-defined edema formation phase (*, p < 0.0001 Mann Whitney). RNA quantification (B) and protein quantification with western-blot (C) confirmed a much stronger increase in the expression of AQP4 during the MRI-defined edema resolution compared to the MRI-defined edema formation phase (*, p < 0.05 Mann Whitney). The inset in (B) shows the area of the tissue micro-dissection. A tissue block of 3 mm was cut around the injection site. Within the block, samples from the injured and contralateral sides were obtained using a 3-mm-core unipunch (right and left shaded circles). In (C), a representative western blot shows the strong increase of AQP4 at 14 and 20 dpi, while actin, which was used to control loading variations, was stable.
Figure 6
Figure 6
MRI/histological correspondence during the build-up phase of edema. A representative rat examined at 1 dpi is shown. (A) A large hypersignal area was seen on the T2WI (dotted line) with high ADC values (dotted line, ADC = 1377 μm2/s as opposed to 1039 μm2/s in the symmetric contralateral area), indicating increased water content. A slight midline shift resulted from the cerebral edema (dotted arrows on T2WI). (B) The corresponding histological sections (low magnification, with white boxes indicating higher magnification positions) showed an increase in AQP4 immunoreactivity in the MRI-defined edematous area, with staining located around the capillaries (arrows) and larger vessels (arrowhead) at the BBB level. AQP4 staining in the symmetric contralateral area is fainter around the capillaries (arrows). (C) Double labeling of GFAP (Alexa 488, green) and AQP4 (CY3, red), examined using confocal microscopy confirmed the perivascular location of AQP4 on astrocyte endfeet surrounding capillaries (arrows) without any AQP4 on the astrocyte body.
Figure 7
Figure 7
MRI/histological correspondence during the resolution phase of edema. A representative rat examined at 20 dpi is shown. (A) MRI showed a small cavitation at the site of the injection with a cerebrospinal-fluid-like signal on the T2WI (arrow) and ADC map, while no peripheral edema was anymore visible along the upper part of the internal capsule (dotted line, ADC = 889 μm2/s as opposed to 852 μm2/s in the symmetric contralateral area). (B) The corresponding histological sections (low magnification, with white boxes indicating higher magnification positions) showed a marked increase in AQP4 immunoreactivity, with staining located around the vessels (arrowhead) and with a fibrillary pattern corresponding to staining on the entire astrocyte membrane in a gliotic area (arrows). The staining in the symmetric contralateral area is more faint and only around capillaries. (C) Double labeling of GFAP (Alexa 488, green) and AQP4 (CY3, red), examined using confocal microscopy, confirmed AQP4 localization over the entire membrane of hypertrophic astrocytes expressing high levels of GFAP and not just around vessels (arrowhead). Double arrows show AQP4 staining along an astrocyte process and dotted arrows show AQP4 staining along an astrocyte cell body.
Figure 8
Figure 8
Suggested model for interstitial edema pathophysiology. The edema build-up phase results from high BBB permeability while AQP4 expression is not yet highly upregulated, resulting in insufficient routes for water elimination. After the time lag of AQP4 expression, edema resolution results from the conjunction of BBB restoration and subsequent significant AQP4 upregulation over the entire astrocyte membrane. Transition phases likely exist between the two extremes.
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