Table 2.

Summary of some published work highlighting the presence of edema in hyperammonemia in-vitro and ex-vivo HE models

Subject	Type of measurements	Method	Comments	Ref
Rat - hyperammonemia - NH4Cl (5 and 10 mM)	In-vitro: primary cultures of astrocytes from neonatal cerebral cortex	Light and electron microscopy	Initial response of astrocytes - of hypertrophy (compensatory) followed by degenerative changes Light microscopy - increased cytoplasmic basophilia, cytoplasmic granularity, vacuolization, formation of dense bodies, and eventual cellular disintegration - Alzheimer type-II-like cells were found in areas adjacent to necrosis Electron microscopy - astrocyte treated for 1 d with 5 mM ammonia shows prominent filamentous dense bodies, loss of glial filaments and occasional vacuoles	Norenberg (1987)
Guinea pig (weight 300–350 g) and rat (weight 220–230 g) - hyperammonemia - NH4Cl (5 and 10 mM)	Ex-vivo: guinea pig and rat cerebellar slices (0.4 mm)	final wet weight vs. initial wet weight of the slices - measure of the increase of water uptake	- guinea pig: brain slices swelling too small to be significant - NH4Cl (5 mM and 10 mM) - rat: significant swelling of the brain slices at NH4Cl 10 mM - the outward flow of K+ was greater than the net inward flow of${\mathit{NH}}_4^{+}$- could not be explained as simply due to an exchange - increased the tissue content of Na+ and Cl− - presumably enhanced water uptake occurs in the glia as the neurons undergo little swelling	Benjamin et al. (1978)
Rat - hyperammonemia - NH4Cl (2, 5, and 10 mM)	In-vitro: primary cultures of astrocytes from neonatal cerebral cortex	Methyl-D-glucose, 3-O-[Methyl-14 C] and [3 H]-3-O-Methylglucose method -${\mathit{\beta }}^{-}$radiation	− 5 mM NH4Cl − 12% ↑ in astrocytes volume after 4 days of treatment (p < 0.02). − 10 mM NH4Cl - one day of treatment: 11 + 1.4% ↑ (p < 0.001) while treatment for 4 days resulted in a 29 ± 3.0% ↑ (p < 0.0001) in astrocytic water space - after 3 days ammonia-induced swelling was still reversible in normal culture media for 1 day - ammonia ↑ lead to astrocyte swelling and may contribute to the brain edema in fulminant liver failure - of note: cells maintained in fetal calf serum did show some swelling upon treatment with ammonia, the effect was smaller and not as consistent as that seen in horse serum-treated cells − 5 mM NH4Cl co-treatment with 0.1 mM aspartate (3 days) - suppressed the ammonia-induced swelling of astrocytes ~ 68% − 1 mM ornithine had no significant effect on the ammonia-induced swelling of astrocytes	Norenberg et al. (1991), Murthy and Norenberg  (2002)
Rat - hyperammonemia - NH4Cl (5 mM) + antioxidants (SOD, CAT, vit. E) and MAPKs inhibitors SB239063 (an inhibitor of p38-MAPKs), and SP600125 (an inhibitor of c-Jun N-terminal kinase, JNK)	In-vitro: primary cultures of astrocytes from neonatal cerebral cortex	Western blots Immunofluorescence of NFκB [3 H]-3-O-Methylglucose method -${\mathit{\beta }}^{-}$radiation Fluorescence spectroscopy	- increased astrocyte swelling by 40.2% (p < 0.05, compared to control) - aminoguanidine (500 µM) partially prevented astrocyte swelling, while 1 mM completely blocked it - activation of NFκB after 12 h exposure to ammonia (5 mM) - inhibitors of NFκB, BAY 11-7082 (5 µM) and SN-50 (3 µM) (76.3 and 66.8%, respectively, as compared to control, p < 0.05) diminished NFκB activation by ammonia - SOD (25 units/ml) and vit. E (100 µM) diminished ammonia induced NFκB activation (69.7, 59.8 and 55.0% respectively, p < 0.05) - ammonia-induced astrocyte swelling was inhibited by the NFκB inhibitors BAY 11-7082 and SN-50 - MAPKs inhibitors attenuate NFκB activation by ammonia - NFκB inhibitors diminish ammonia-induced astrocyte swelling - significant role of oxidative stress and MAPKs in NFκB activation - activation of NFκB was associated with increased iNOS protein expression and NO ↑, and these changes were blocked by BAY 11-7082, an inhibitor of NFκB	Sinke et al. (2008)
Rat - hyperammonemia - NH4Cl (5 mM) + cytokines - TNF-α, IL-1b, IL-6 and IFN-g, all 10 ng/ml each, either individually or in combination and NF-kB inhibitor (BAY 11-7082)	In-vitro: primary cultures of astrocytes from neonatal cerebral cortex	[3 H]-3-O-Methylglucose method -${\mathit{\beta }}^{-}$radiation Western blots	- key role of NF-kB in the mechanism of ammonia-induced astrocyte swelling - swelling was increased by ammonia (43%) and by cytokines (37%) at 24 h - co-treatment with cytokines and ammonia showed no additional swelling − 24 h ammonia pretreatment followed by additional 24 h exposure to cytokines → a marked increase in astrocyte swelling ~ 129% - treatment with ammonia or cytokines alone also activated NF-kB (80–130%) − 24 h ammonia pretreatment followed by additional 24 h exposure to cytokines → a marked activation of NF-kB (428%) - NF-kB inhibitor (BAY 11-7082), completely blocked the astrocyte swelling in cultures pre-treated with ammonia and followed by the addition of cytokines	Rao et al. (2010)
Rat - hyperammonemia - NH4Cl or NH4CH3CO2 (2, 5 and 10 mM, 1–10 days)	In-vitro: Primary cultures of astrocytes from neonatal cerebral cortex	Light and electron microscopy	Acute ammonia intoxication Light microscopy - increase in basophilia - prominent cytoplasmic processes - increased cytoplasmic granularity and microvacuolization - increase in nucleolar/nuclear ratio - fragmentation of cytoplasmic processes - formation of dense bodies - cellular disintegration Electron microscopy - disaggregation of polyribosomal clusters - loss of glia filaments - RER and SER ↑ - mitochondria number and length↑ - mitochondria swelling with intercristal space widening - increased cytoplasmic granularity and microvacuolization	Gregorios et al. (1985a, b)
Rat - hypoosmotic exposure (205 mosmol/L)	In-vitro: cultured rat astrocytes - from the cerebral hemispheres of newborn rats	Western blot	- induces MAP kinase activation - important role of extracellular calcium in the osmo-signaling pathway upstream of the MAP kinases - sustained elevation of the intracellular Ca2+ concentration - entry from the extracellular space - hypoosmotic astrocyte swelling activates MAP-kinases in a Ras/Raf-dependent and PI3-kinase-dependent way which is initiated by a swelling-induced Ca2+ signal	Schliess et al. (1996), Häussinger et al. (1997)
Rat - hyperammonemia - NH4Cl (1, 5 and 10 mM)	Ex-vivo: brain slices (cortex)	Dry-weight technique Light microscopy	- initial water content 83.03 ± 0.54% (control) → 86.25 ± 1.16% (5 mM) → 88 ± 0.62% (10 mM) - swelling - water accumulation ↑ with ammonia concentration ↑ - increased intracellular Na+ and decreased K+ content - decrease in the space of distribution of inulin was seen at the 10 mM concentration, suggesting intracellular water accumulation - spongiform change of the neuropil - enlarged astrocytic nuclei, redistribution of chromatin, and a clear nucleoplasm	Ganz et al.  (1989)
Rat / mouse – hypoosmolality, hyperammonemia - NH4Cl (5 mM)	In-vitro: cultured rat astrocytes - from the cortices of newborn rats Ex-vivo: brain slices (cortex) from male mice (adult)	Immunostaining Fluorescence microscopy Western Blot	Acute ammonia intoxication - hypoosmotic (205 mosmol/L) swelling of cultured astrocytes induced a rapid generation of ROS - ammonia, similar to hypoosmolality, induced a rapid astrocyte swelling - ROS production by ammonia is accompanied by a rapid astrocyte swelling, which is reversible upon removal of ammonia - close interrelation between astrocyte swelling and oxidative stress	Reinehr et al.  (2007)
Rat / mouse – hyperammonemia - NH4Cl (5 mM)	In-vitro: cultured rat astrocytes - from the cortices of cerebral hemispheres of newborn rats	IHC - confocal laser-scanning microscopy North-Western and Slot-Blot Analysis - isolated RNA	Acute ammonia intoxication - ammonia and hypoosmotic swelling increased RNA oxidation - ammonia-induced RNA oxidation is reversible in-vivo	Gorg et al.  (2008)
Rat / mouse – primary astrocytes / neurons - hyperammonemia - NH4Cl (5 mM) Rat - hyperammonemia (weight 280 ± 6 g) - NH4CH3CO2 (4.5 mM)	In-vitro: cultured rat astrocytes - from the cortices of cerebral hemispheres of newborn Wistar rats co-cultured with hippocampal neurons from embryonic C57BL/6Jmice Ex-vivo: brain slices (cortex)	Fluorescence and electron microscopy	In-vitro: - moderately increased concentrations of NH4Cl induce autophagy while a high concentration ~ 5  mM / hyperammonemia inhibits autophagic flux in primary astrocytes − 5mM NH4Cl → 5-fold increase in the number of autophagosomes/autolysosomes compared to the water-treated control - inhibition of autophagy is largely mediated by changes in intracellular and intralysosomal pH - ammonia-induced inhibition of autophagy occurs in a ROS-dependent manner - neurons show impairment of autophagy under hyperammonemia Ex-vivo: -astrocytes and neurons autophagy is impaired by hyperammonemia - varying degree in certain brain regions	Lu et al.  (2019)
Mouse – hyperammonemia - NH4CH3CO2 (1–10 mM)	In-vitro: post-natal organotypic slice cultures – forebrain	Optical coherence tomography (OCT) - of cultured slices Confocal microscopy Immunoblotting	- ammonia for 3 days → evidence of tissue edema that is linked with astrocytic swelling - no significant changes in slice area were detected over 3 days at ammonia concentrations up to 10 mM - NH4CH3CO2 10 mM resulted in macroscopic tissue swelling, with slice thickness increasing ~ 30%, treated: 441 ± 40 μm vs. control: 345 ± 33 μm - volume of GFAP-immunoreactivity was used as a marker of astrocyte cytoplasmic volume - the fractional volume of cortex occupied by GFAP- immunoreactive voxels progressively and significantly increased at concentrations of 1–10 mM of NH4CH3CO2 - NH4CH3CO2 10 mM resulted in 8% increase in volume of GFAP-immunoreactivity - no change in astrocytes number - neurons somatic diameter of control ranged from 10–15 μm vs. 20 μm after treatment with NH4CH3CO2 10 mM associated with large swollen nuclei	Back et al.  (2011)
C6 Glioma Cell line (AmericanType Culture Collection (Rockville, MD)) -hyperammonemia - NH4Cl (5 mM) + resveratrol (100 µM)	In-vitro: astroglia cells	Optical microscopy	- ammonia induced astrocyte swelling / cell body retraction, and resveratrol prevented this effect - ammonia induced increased expression of cytokines, the TNF-α, IL-6 and IL-1β, which was partially reversed by resveratrol (TNF-a levels from 148 ± 13% to 109 ± 13%, IL-1β levels from 140 ± 9% to 110 ± 13%, and IL-6 from 135 ± 13% to 115 ± 16%, values expressed as percentage of control)	Bobermin et al. (2012)
Brain cell 3D culture hyperammonemia - NH4Cl (5 mM) + creatine (1 mM)	In-vitro: mixed-cell aggregates – from mechanically dissociated telencephalon of 16-day rat embryos	Optical microscopy	- ammonia induced astrocyte swelling, and creatine prevents this effect - NH4Cl alters AGAT, GAMT and SLC6A8 expression in glial cells but not in neurons - increased GAMT but repressed SLC6A8 mRNA expression in oligodendrocytes - creatine co-treatment under NH4Cl exposure represses AGAT and SLC6A8 in swollen astrocytes - decrease of endogenous synthesis of creatine under NH4Cl exposure - ammonia exposure impaired axonal growth in developing mixed cell aggregates - creatine prevented ammonia-induced axonal growth impairment in developing mixed cell aggregates - creatine did not prevent the ammonia-induced axonal growth impairment in developing neuron-enriched aggregates - neuronal fiber growth impairment under ammonium exposure, together with a decrease in medium size neurofilaments (NF-M) - in mature aggregates ammonia exposure does not alter axonal morphology → difference in vulnerability of the developing and adult brain - no significant effect was found for oligodendrocytes	Braissant et al.  (2002)