Why does ischemia cause edema

Abstract Brain edema is a life-threatening complication of cerebral infarction. Publication types Research Support, U. Gov't, Non-P. Cytotoxic cerebral edema refers to a type of cerebral edema , most commonly seen in cerebral ischemia , in which extracellular water passes into cells, resulting in their swelling.

The term is frequently used in clinical practice to denote the combination of true cytotoxic edema and ionic cerebral edema. As the pathophysiology of these two types of edema is different, as is their imaging, they are discussed separately. The remainder of this article is concerned with true cytotoxic edema, also known as cellular edema 8.

This, in turn, results in cellular swelling and a reduction in the extracellular volume which are the primary reasons for increased restricted diffusion on MRI. This intracellular edema mainly affects grey matter but also involves the white matter as astrocytes are also involved. In contrast to vasogenic cerebral edema , in which the blood brain barrier is compromised, cytotoxic edema does not involve endothelial dysfunction or changes in capillary permeability.

In true isolated cytotoxic edema little change is evident on CT as a mere redistribution of water from extracellular to intracellular compartments does not result in attenuation changes.

The changes colloquially ascribed to 'cytotoxic edema' are in fact mostly due to ionic edema and are described separately. This is why brain CT is often normal in patients with an acute ischemic stroke.

As cytotoxic edema represents the redistribution of water from extracellular to intracellular compartments, without a change in local constituents it stands to reason that no T1 or T2 changes are evident. As is the case with CT, the changes colloquially ascribed to 'cytotoxic edema' are in fact mostly due to ionic edema and are described separately. The one sequence which is able to identify cytotoxic edema, and was thus responsible for a revolution in the imaging of acute ischemic stroke, is diffusion weighted imaging DWI.

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Zuo, X. Keywords: ischemia stroke, cerebral edema, molecular mechanism, drug therapy, preclinical drug evaluation, clinical trial. Aging Neurosci. The use, distribution or reproduction in other forums is permitted, provided the original author s and the copyright owner s are credited and that the original publication in this journal is cited, in accordance with accepted academic practice.

No use, distribution or reproduction is permitted which does not comply with these terms. Introduction Stroke is the leading cause of death worldwide Feigin et al. Mechanisms of Cerebral Edema Cerebral edema after an ischemic stroke includes cytotoxic edema, ionic edema, and vasogenic edema Liebeskind et al. Table 2. The mechanisms of cerebral edema and the various anti-edema agents.

Inset: contralateral non-affected vessel. Since arteries, capillaries and veins display distinct functional and morphological differences [ 5 ], we also addressed different segments of the vascular tree.

Therefore, we applied double immunofluorescence labeling of laminin to reliably identify vessels irrespective of their positions in the vascular tree in combination with SMA to identify arteries [ 23 , 34 ].

Here, FITC-albumin extravasations displayed comparable fluorescence intensities along arteries, capillaries and veins Fig. Although individual extravasations were found to be less wide-spread at the capillary level, the overall contribution per field of view appeared to be slightly higher for capillaries compared to arteries and veins.

In line with the results shown in Fig. Importantly, arterial and capillary endothelial cells exhibit comparable scores of ultrastructural damage in the ischemia-affected striatum and the cortex.

As arteries are known to face much higher pressures and blood flow velocities [ 43 , 62 ], we also applied electron microscopy to investigate whether the ultrastructural alterations are more pronounced in arterial endothelial cells compared to adjacent capillary vessels. However, in each of the applied models, the mean scores of ultrastructural damage did not differ when comparing arterial and adjacent capillary endothelial cells.

Importantly, the astrocytic expression of Aqp4 water channels is restricted to the vascular surface and the vascular basement membrane under physiological conditions. In shorter periods of ischemia, these alterations were not observed. In contralateral control regions ctrl, inset Aqp4 expression is highly polarized and confined to astrocytic endfeet directly adjacent to the vascular basement membrane. Given the fact that Cx43 hemi-channels have been suggested to mediate a cellular edema in the setting of stroke, we further investigated the Cx43 expression in ischemia-affected vessels.

Further, we addressed the question whether or not the described vascular alterations involving BBB breakdown for FITC-albumin are restricted to the ischemic core, or potentially involve salvageable tissue of the ischemic penumbra [ 59 ].

As an ischemia-sensitive marker, MAP2 immunoreactivity is decreased in ischemic areas of the ischemic core and the surrounding penumbral areas [ 31 , 55 ]. In contrast, HSP70 is up-regulated in ischemia-affected neurons of the penumbra, but not in the ischemic core [ 59 ]. In line, FITC-albumin extravasation is not confined to the ischemic core, but is also detectable in penumbral areas characterized by a neuronal upregulation of HSP70 Fig.

Here, neuronal HSP70 expression was predominantly observed in cortical areas. Of note, FITC-albumin extravasations are not restricted to penumbral areas, but are also found in striatal areas of the presumed ischemic core, which are lacking a selective HSP70 up-regulation in neurons. Further, we investigated whether areas of the ischemic core and the adjacent penumbra contribute differently to BBB breakdown.

For this purpose striatal and cortical areas lacking neuronal HSP70 expression were compared with cortical areas of neuronal HSP70 expression, the latter of which represent penumbral areas. In these areas, the mean fluorescence intensity as well as the area of extravasated FITC-albumin was measured in different fields of view per section and animal. Although penumbral areas of the cerebral cortex revealed a trend towards less pronounced fluorescence intensities and less wide-spread FITC-albumin distributions per field of view, the differences failed to reach statistical significance Fig.

Although BBB breakdown has been predominantly linked to a failure of TJ complexes to seal the interendothelial cleft [ 17 , 29 , 58 ], increasing evidence suggest a transendothelial leakage mechanism to underlie impaired BBB function in the setting of stroke [ 11 , 22 ].

In this context, our group demonstrated severe structural degenerations of the endothelial layer, including loss of endothelial integrity and partial detachment of the endothelium from the vascular wall in different models of focal cerebral ischemia [ 33 , 34 , 35 ].

These alterations of the vasculature are therefore likely to represent morphological features of the ischemia-affected NVU harboring the risk for stroke-related complications like intracerebral bleeding, especially after therapeutic vessel recanalization [ 56 , 64 ]. Therefore, the present study was aimed to investigate ischemia-induced affections of the endothelial layer in early stages after stroke.

To assure, that the described analyses indeed refer to ischemia-affected areas showing BBB breakdown, we applied the established permeability marker FITC-albumin. This reagent offers the advantage of a reliable detectability in sections used for immunofluorescence microscopy and upon DAB staining, even in sections for light and electron microscopy [ 33 , 34 ]. Furthermore, the use of FITC-albumin as a permeability marker is facilitated by its outstanding fixability, thereby allowing reliable detection in the tissue, even after extensive steps of rinsing.

It also proved to offer an excellent antigenicity allowing 5 times higher concentrations of glutaraldehyde in the fixative to provide optimal preservation of the ultrastructure compared to standard protocols for immunoelectron microscopy.

Thereby the risk of mechanical or peroxidase-related artifacts can be further reduced. Although BBB permeability profiles for ions and dextrans of smaller molecular weight may not necessarily comply with the applied FITC-albumin, the latter is also of clinical interest as the extravasation of albumin is known to promote epileptic seizures as a typical complication of stroke [ 28 , 37 ].

Moreover, especially dextran tracers of lower molecular weight are reported to produce false negative results, as they are easily washed out of the respective tissue [ 26 ]. In line with other reports on TJ-independent mechanisms of BBB breakdown [ 30 , 52 , 57 ], the present analyses reveal that claudin 5- and occludin-positive TJ strands remain detectable in each of the investigated time points Fig. Although slightly decreased protein levels of occludin were found in ischemia-affected striatal areas, these alterations could not be captured at the level of fluorescence microscopy.

Importantly, these findings are consistent with our previous observations from the embolic, permanent and transient MCAO models at the h time point after ischemia onset in mice and rats [ 33 , 35 ], which has been shown to coincide with the peak of edema formation following stroke [ 53 ].

However, since immunolabeling of TJ proteins is often used to evaluate BBB integrity, the presented findings clearly demonstrate that detection of TJ markers alone cannot be correlated with BBB function. In contrast, at the level of fluorescence microscopy lectin staining with I-B4 proved to detect gaps and discontinuities of lectin binding sites at the endothelial surface in vessels showing BBB breakdown Fig. Here, electron microscopy revealed severe degenerations of the endothelial layer, which were detectable in each of the applied time points.

These alterations include an endothelial edema, endothelial uptake of FITC-albumin, endothelial disintegration with leakage of the tracer into the parenchyma and extravasation of erythrocytes.

Importantly, the severe structural alterations of the endothelium described at the level of electron microscopy can be supported by the endothelial staining using I-B4. Here, the discontinuous endothelial staining indicating spatially reduced lectin binding sites may likely relate to the impaired endothelial integrity observed at the level of electron microscopy.

In light of the higher blood pressure within arterial segments compared to capillary or venous vessels [ 43 , 62 ], it is also remarkable that capillaries and arteries exhibit comparable scores of vascular damage throughout the applied models. While the capillary segments exhibited a slightly higher contribution to the FITC-albumin extravasations Fig. In this context, the potential influence of signaling cascades from adjacent cell types cells can also be considered [ 2 , 7 ].

However, larger extra- and intracerebral arteries are likely to be differently affected by catheter-based mechanical manipulations in the clinical setting of thrombectomy. Noteworthy, the detection of FITC-albumin loaded endothelial vesicles and caveolae in cells showing an endothelial edema Fig. In parallel, further swelling of the endothelial cell is likely to lead to the disruption of the plasma membrane, which also facilitates uptake of the tracer, finally leading to disintegration and partial loss of the endothelial layer.

This concept finds support in studies suggesting Cx43 hemichannels to play a pivotal role in ischemia-mediated cell swelling [ 8 , 9 , 10 , 20 ]. Since Cx43 is also expressed in the brain parenchyma, the slight decrease of the Cx43 protein level in ischemia affected areas is likely to refer to non-vascular structures. However, since pharmacological blocking of Cx43 hemichannels is shown to increase neuronal survival an adjuvant treatment may also turn out to protect the endothelial layer [ 20 ].

Further, astrocytes have been shown to critically impact on the ischemia-associated edema formation which involves Aqp4 water channels [ 61 ]. Although the concept of an ischemic core and a shell-like penumbra originally refers to levels of blood flow ensuring neuronal survival [ 3 ], alterations of the cellular metabolism can also be considered to characterize ischemic areas and peri-infarct regions.

Further, an altered expression of ischemia sensitive markers such as MAP2 can be ascribed to reversibly affected tissue representing the ischemic penumbra, as well [ 31 , 41 , 55 ].

However, the thresholds of cerebral blood flow ensuring neuronal survival in penumbral areas [ 3 ] may not necessarily be applicable to cells of the ischemia-affected vessels, themselves. Therefore, the potential reversibility of the described endothelial alterations remains to be investigated by future studies. While severe endothelial alterations with lost cellular integrity are likely to be irreversible, the observed endothelial edema may turn out to be reversible upon restored cerebral perfusion.

Despite of the descriptive study design, we here for the first time provide evidence for severe structural alterations of the endothelial layer in early time points after ischemia induction. In this process, the vascular degeneration may ultimately increase the risk of hemorrhagic transformation and intracranial bleeding following therapeutic restoration of the cerebral blood flow and intravascular blood pressure [ 56 , 64 ].

Deciphering the pathophysiology of the ischemia-affected NVU including endothelial dysfunction was therefore rated as high priority for stroke research [ 15 , 44 ], while further insights will hopefully allow the development of adjuvant therapies which may help to extend the therapeutic time window and to protect BBB function in the setting of stroke.

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