Table 4.

Summary of peritumoral edema measures

Edema measure	Description
Histology	We included studies that used any type of histopathological technique. These included “simple” staining and light microscopy, immune-staining and light-microscopy as well as scanning- and transmission electromicrography techniques. Factors such as distance between cells can then be used as a measure of edema. Histopathological techniques require post-mortem tissue for analysis and therefore allow only “snapshot” sampling of edema. Histopathological techniques were frequently used concurrently with extravasation of immune-stainable substances not normally found in brain tissue, either endogenous (albumin) or exogenous (horseradish peroxidase) to provide some measure of “leakiness” of cerebral vasculature
Brain water content (BWC)	Brain water content studies predominantly use the difference between wet and dried weight of an aliquot of brain tissue to estimate the water content of the tissue. Tissue is harvested post-mortem, weighed and then dried according to protocol (usually in an oven) and weighed again afterwards. This method cannot distinguish between increased extracellular fluid, as seen in peritumoral vasogenic edema, and increased water content e.g. due to cytotoxic edema
Extravasation studies	Extravasation studies include any studies that endeavor to establish the concentration of a chemical that is not normally present in brain tissue but occurs either naturally in blood, or is injected. The most commonly used substances were dyes such as Evans blue, exogenous proteins like horseradish peroxidase and endogenous proteins like albumin. The presence or concentration of these can then be detected in tissue post-mortem by a variety of techniques such as immunostaining microscopy, Western blotting and spectroscopy. This technique is an indirect measure of blood–brain barrier “leakiness” rather than edema, but the former is often used by investigators as a proxy for the latter
Imaging	Imaging can be used to investigate animals in vivo for radiographic appearances suggestive of tumor and/or peritumoral edema. The most common imaging modality is MRI where one would expect areas of high T2/FLAIR and low T1 signal relative to surrounding grey/white matter around the tumor to correspond to edema. However, imaging interpretation can be complex, especially in tumors that form complex heterogeneous masses which makes interpretation of imaging more difficult. MRI scans are considerably more expensive than the other techniques outlined here
Symptomatology	Assessing symptomatology in animals can be done in a number of ways; the simplest is to use a measure of mortality e.g. the time from the tumor instigation until death. More in-depth techniques have been used in some studies, for instance, observing for unilateral weakness or pupillary mydriasis (signs of considerable localizing mass effect) or observing for behavioral change (failure to feed, failure to groom). This has the benefit of measuring what would likely constitute a primary outcome in any human trial (morbidity/mortality) and can be done cheaply and repeatedly over time. It should be noted, however, that these measures do not differentiate between tumor growth and isolated tumor edema
Electrolyte composition assay	Similar to BWC, an aliquot of brain tissue is homogenized and ashened before quantifying the electrolyte content for instance with liquid chromatography. This technique is occasionally seen reported together with BWC, with which it shares significant methodological overlap
Permeability studies	We included in this category studies that used infusion of substances such as radio-labelled small molecules like aminoisobutyric acid, to allow calculation of a permeability constant. This is a more formal quantification of the permeability of brain vasculature to that particular substance than can be obtained by simply injecting a bolus of a substance as with extravasation studies. These techniques require sacrificing the animal