Age and Sex in the Development of Hepatic Encephalopathy: Role of Alcohol.

Hepatic encephalopathy (HE) is a neurological condition linked to liver failure. Acute HE (Type A) occurs with acute liver failure, while chronic HE (Type C) is tied to cirrhosis and portal hypertension. HE treatments lag due to gaps in understanding its development by gender and age. We studied how sex and age impact HE and its severity with combined liver toxins. Our findings indicate that drug-induced (thioacetamide, TAA) brain edema was more severe in aged males than in young males or young/aged female rats. However, adding alcohol (ethanol, EtOH) worsens TAA's brain edema in both young and aged females, with females experiencing a more severe effect than males. These patterns also apply to Type A HE induced by azoxymethane (AZO) in mice. Similarly, TAA-induced behavioral deficits in Type C HE were milder in young and aged females than in males. Conversely, EtOH and TAA in young/aged males led to severe brain edema and fatality without noticeable behavioral changes. TAA metabolism was slower in aged males than in young or middle-aged rats. When TAA-treated aged male rats received EtOH, there was a slow and sustained plasma level of thioacetamide sulfoxide (TASO). This suggests that with EtOH, TAA-induced HE is more severe in aged males. TAA metabolism was similar in young, middle-aged, and aged female rats. However, with EtOH, young and aged females experience more severe drug-induced HE as compared to middle-aged adult rats. These findings strongly suggest that gender and age play a role in the severity of HE development and that the presence of one or more liver toxins may aggravate the severity of the disease progression.

Multi-Organ Histopathological Changes in a Mouse Hepatitis Virus Model of COVID-19.

Infection with SARS-CoV-2, the virus responsible for the global COVID-19 pandemic, causes a respiratory illness that can severely impact other organ systems and is possibly precipitated by cytokine storm, septic shock, thrombosis, and oxidative stress. SARS-CoV-2 infected individuals may be asymptomatic or may experience mild, moderate, or severe symptoms with or without pneumonia. The mechanisms by which SARS-CoV-2 infects humans are largely unknown. Mouse hepatitis virus 1 (MHV-1)-induced infection was used as a highly relevant surrogate animal model for this study. We further characterized this animal model and compared it with SARS-CoV-2 infection in humans. MHV-1 inoculated mice displayed death as well as weight loss, as reported earlier. We showed that MHV-1-infected mice at days 7-8 exhibit severe lung inflammation, peribronchiolar interstitial infiltration, bronchiolar epithelial cell necrosis and intra-alveolar necrotic debris, alveolar exudation (surrounding alveolar walls have capillaries that are dilated and filled with red blood cells), mononuclear cell infiltration, hyaline membrane formation, the presence of hemosiderin-laden macrophages, and interstitial edema. When compared to uninfected mice, the infected mice showed severe liver vascular congestion, luminal thrombosis of portal and sinusoidal vessels, hepatocyte degeneration, cell necrosis, and hemorrhagic changes. Proximal and distal tubular necrosis, hemorrhage in interstitial tissue, and the vacuolation of renal tubules were observed. The heart showed severe interstitial edema, vascular congestion, and dilation, as well as red blood cell extravasation into the interstitium. Upon examination of the MHV-1 infected mice brain, we observed congested blood vessels, perivascular cavitation, cortical pericellular halos, vacuolation of neuropils, darkly stained nuclei, pyknotic nuclei, and associated vacuolation of the neuropil in the cortex, as well as acute eosinophilic necrosis and necrotic neurons with fragmented nuclei and vacuolation in the hippocampus. Our findings suggest that the widespread thrombotic events observed in the surrogate animal model for SARS-CoV-2 mimic the reported findings in SARS-CoV-2 infected humans, representing a highly relevant and safe animal model for the study of the pathophysiologic mechanisms of SARS-CoV-2 for potential therapeutic interventions.

Alpha-synuclein increases in rodent and human spinal cord injury and promotes inflammation and tissue loss.

Synucleinopathies are neurodegenerative diseases in which α-synuclein protein accumulates in neurons and glia. In these diseases, α-synuclein forms dense intracellular aggregates that are disease hallmarks and actively contribute to tissue pathology. Interestingly, many pathological mechanisms, including iron accumulation and lipid peroxidation, are shared between classical synucleinopathies such as Alzheimer's disease, Parkinson's disease and traumatic spinal cord injury (SCI). However, to date, no studies have determined if α-synuclein accumulation occurs after human SCI. To examine this, cross-sections from injured and non-injured human spinal cords were immunolabeled for α-synuclein. This showed robust α-synuclein accumulation in profiles resembling axons and astrocytes in tissue surrounding the injury, revealing that α-synuclein markedly aggregates in traumatically injured human spinal cords. We also detected significant iron deposition in the injury site, a known catalyst for α-synuclein aggregation. Next a rodent SCI model mimicking the histological features of human SCI revealed aggregates and structurally altered monomers of α-synuclein are present after SCI. To determine if α-synuclein exacerbates SCI pathology, α-synuclein knockout mice were tested. Compared to wild type mice, α-synuclein knockout mice had significantly more spared axons and neurons and lower pro-inflammatory mediators, macrophage accumulation, and iron deposition in the injured spinal cord. Interestingly, locomotor analysis revealed that α-synuclein may be essential for dopamine-mediated hindlimb function after SCI. Collectively, the marked upregulation and long-lasting accumulation of α-synuclein and iron suggests that SCI may fit within the family of synucleinopathies and offer new therapeutic targets for promoting neuron preservation and improving function after spinal trauma.

Age as a determinant of inflammatory response and survival of glia and axons after human traumatic spinal cord injury.

Despite the shift in the demographics of traumatic spinal cord injury (SCI) with increased proportion of injuries in the elderly, little is known on the potential effects of old age on the pathobiology of SCI. Since there is an assumption that age adversely affects neural response to SCI, this study examines the clinically relevant question on whether age is a key determinant of inflammatory response, oligodendroglial apoptosis and axonal survival after traumatic SCI. This unique study includes post-mortem spinal cord tissue from 64 cases of SCI (at cervical or high-thoracic levels) and 38 control cases without CNS injury. Each group was subdivided into subgroups of younger and elderly individuals (65 years of age or older at the SCI onset). The results of this study indicate that age at the SCI onset does not adversely affect the cellular inflammatory response to, oligodendroglial apoptosis and axonal survival after SCI. These results support the conclusion that elderly individuals have similar neurobiological responses to SCI as younger people and, hence, treatment decisions should be based on an assessment of the individual patient and not an arbitrary assumption that "advanced age" should exclude patients with an acute SCI from access to advanced care and translational therapies.

Additive Effect of Resveratrol on Astrocyte Swelling Post-exposure to Ammonia, Ischemia and Trauma In Vitro.

Swelling of astrocytes represents a major component of the brain edema associated with many neurological conditions, including acute hepatic encephalopathy (AHE), traumatic brain injury (TBI) and ischemia. It has previously been reported that exposure of cultured astrocytes to ammonia (a factor strongly implicated in the pathogenesis of AHE), oxygen/glucose deprivation, or to direct mechanical trauma results in an increase in cell swelling. Since dietary polyphenols have been shown to exert a protective effect against cell injury, we examined whether resveratrol (RSV, 3,5,4'-trihydroxy-trans-stilbene, a stilbenoid phenol), has a protective effect on astrocyte swelling following its exposure to ammonia, oxygen-glucose deprivation (OGD), or trauma in vitro. Ammonia increased astrocyte swelling, and pre- or post-treatment of astrocytes with 10 and 25 µM RSV displayed an additive effect, while 5 µM did not prevent the effect of ammonia. However, pre-treatment of astrocytes with 25 µM RSV slightly, but significantly, reduced the trauma-induced astrocyte swelling at earlier time points (3 h), while post-treatment had no significant effect on the trauma-induced cell swelling at the 3 h time point. Instead, pre- or post-treatment of astrocytes with 25 µM RSV had an additive effect on trauma-induced astrocyte swelling. Further, pre- or post-treatment of astrocytes with 5 or 10 µM RSV had no significant effect on trauma-induced astrocyte swelling. When 5 or 10 µM RSV were added prior to, or during the process of OGD, as well as post-OGD, it caused a slight, but not statistically significant decline in cell swelling. However, when 25 µM RSV was added during the process of OGD, as well as after the cells were returned to normal condition (90 min period), such treatment showed an additive effect on the OGD-induced astrocyte swelling. Noteworthy, a higher concentration of RSV (25 µM) exhibited an additive effect on levels of phosphorylated forms of ERK1/2, and p38MAPK, as well as an increased activity of the Na+-K+-Cl- co-transporter-1 (NKCC1), factors known to induce astrocytes swelling, when the cells were treated with ammonia or after trauma or ischemia. Further, inhibition of ERK1/2, and p38MAPK diminished the RSV-induced exacerbation of cell swelling post-ammonia, trauma and OGD treatment. These findings strongly suggest that treatment of cultured astrocytes with RSV enhanced the ammonia, ischemia and trauma-induced cell swelling, likely through the exacerbation of intercellular signaling kinases and ion transporters. Accordingly, caution should be exercised when using RSV for the treatment of these neurological conditions, especially when brain edema is also suspected.

Frizzled 1 and Wnt1 as new potential therapeutic targets in the traumatically injured spinal cord.

Despite the experimental evidence pointing to a significant role of the Wnt family of proteins in physiological and pathological rodent spinal cord functioning, its potential relevance in the healthy and traumatically injured human spinal cord as well as its therapeutic potential in spinal cord injury (SCI) are still poorly understood. To get further insight into these interesting issues, we first demonstrated by quantitative Real-Time PCR and simple immunohistochemistry that detectable mRNA expression of most Wnt components, as well as protein expression of all known Wnt receptors, can be found in the healthy human spinal cord, supporting its potential involvement in human spinal cord physiology. Moreover, evaluation of Frizzled (Fz) 1 expression by double immunohistochemistry showed that its spatio-temporal and cellular expression pattern in the traumatically injured human spinal cord is equivalent to that observed in a clinically relevant model of rat SCI and suggests its potential involvement in SCI progression/outcome. Accordingly, we found that long-term lentiviral-mediated overexpression of the Fz1 ligand Wnt1 after rat SCI improves motor functional recovery, increases myelin preservation and neuronal survival, and reduces early astroglial reactivity and NG2+ cell accumulation, highlighting the therapeutic potential of Wnt1 in this neuropathological situation.

Spatio-temporal and Cellular Expression Patterns of PTK7 in the Healthy and Traumatically Injured Rat and Human Spinal Cord.

Despite the emerging role of protein tyrosine kinase 7 (PTK7) as a Wnt co-receptor and the relevant functions of the Wnt family of proteins in spinal cord injury (SCI), the potential involvement of PTK7 in SCI is currently unknown. As a first essential step to shed light on this issue, we evaluated the spatio-temporal and cellular expression patterns of PTK7 in healthy and traumatically injured rat and human spinal cords. In the uninjured rats, PTK7 expression was observed in the ependymal epithelium, endothelial cells, meningeal fibronectin-expressing cells, and specific axonal tracts, but not in microglia, astrocytes, neurons, oligodendrocytes, or NG2+ cells. After rat SCI, the mRNA expression of PTK7 was significantly increased, while its spatio-temporal and cellular protein expression patterns also suffered evident changes in the injured region. Briefly, the expression of PTK7 in the affected areas was observed in axons, reactive astrocytes, NG2+ and fibronectin-expressing cells, and in a subpopulation of reactive microglia/macrophages and blood vessels. Finally, in both healthy and traumatically injured human spinal cords, PTK7 expression pattern was similar to that observed in the rat, although some specific differences were found. In conclusion, we demonstrate for the first time that PTK7 is constitutively expressed in the healthy adult rat and human spinal cord and that its expression pattern clearly varied after rat and human SCI which, to our knowledge, constitutes the first experimental evidence pointing to the potential involvement of this co-receptor in physiological and pathological spinal cord functioning.

Hyperammonemia in Hepatic Encephalopathy.

The precise mechanism underlying the neurotoxicity of Hepatic Encephalopathy (HE) is remains unclear. The dominant view has been that gut-derived nitrogenous toxins are not extracted by the diseased liver and thereby enter the brain. Among the various toxins proposed, the case for ammonia is most compelling. Events that lead to increased levels of blood or brain ammonia have been shown to worsen HE, whereas reducing blood ammonia levels alleviates HE. Clinical, pathological, and biochemical changes observed in HE can be reproduced by increasing blood or brain ammonia levels in experimental animals, while exposure of cultured astrocytes to ammonium salts reproduces the morphological and biochemical findings observed in HE. However, factors other than ammonia have recently been proposed to be involved in the development of HE, including cytokines and other blood and brain immune factors. Moreover, recent studies have questioned the critical role of ammonia in the pathogenesis of HE since blood ammonia levels do not always correlate with the level/severity of encephalopathy. This review summarizes the vital role of ammonia in the pathogenesis of HE in humans, as well as in experimental models of acute and chronic liver failure. It further emphasizes recent advances in the molecular mechanisms involved in the progression of neurological complications that occur in acute and chronic liver failure.

Cells in the adult human spinal cord ependymal region do not proliferate after injury.

In vertebrates that regenerate the injured spinal cord, cells at the ependymal region proliferate and coordinate the formation of bridges between the lesion stumps. In mammals, these cells also proliferate profusely around the central canal after spinal cord injury, although their actual contribution to repair is controversial. In humans, however, the central canal disappears from early childhood in the majority of individuals, being replaced by astrocyte gliosis, ependymocyte clusters, and perivascular pseudo-rosettes. In this human ependymal remnant, cells do not proliferate under normal conditions, but it is not known if they do after a lesion. Here, we studied the human ependymal remnant after traumatic spinal cord injury using samples from 21 individuals with survival times ranging from days to months post-injury. With three different monoclonal antibodies raised against two different proliferation markers (Ki67 and MCM2), we found that the ependymal remnant in adult humans does not proliferate after injury at any time or distance from the lesion. Our results seriously challenge the view of the spinal cord ependymal region as a neurogenic niche in adult humans and suggest that it would not be involved in cell replacement after a lesion. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Neurosarcoidosis presenting as an incidental solitary cranial ring-enhancing lesion.

Isolated neurosarcoidosis without prior systemic symptoms is extremely rare, occurring in approximately 2% of patients with neurosarcoidosis. Due to its rarity, mistakes in diagnosis and treatment occur commonly. We present a case of a 47-year-old female who was found to have an incidentally discovered solitary intracranial lesion that mimicked a high-grade neoplasm, but was later confirmed to be neurosarcoidosis. Incidental solitary neurosarcoid granulomas are difficult to diagnose due to its nonspecific clinical and imaging presentations. Early diagnosis by biopsy in these rare incidental cases remains paramount for adequate treatment.

Human Schwann cells exhibit long-term cell survival, are not tumorigenic and promote repair when transplanted into the contused spinal cord.

The transplantation of rodent Schwann cells (SCs) provides anatomical and functional restitution in a variety of spinal cord injury (SCI) models, supporting the recent translation of SCs to phase 1 clinical trials for human SCI. Whereas human (Hu)SCs have been examined experimentally in a complete SCI transection paradigm, to date the reported behavior of SCs when transplanted after a clinically relevant contusive SCI has been restricted to the use of rodent SCs. Here, in a xenotransplant, contusive SCI paradigm, the survival, biodistribution, proliferation and tumorgenicity as well as host responses to HuSCs, cultured according to a protocol analogous to that developed for clinical application, were investigated. HuSCs persisted within the contused nude rat spinal cord through 6 months after transplantation (longest time examined), exhibited low cell proliferation, displayed no evidence of tumorigenicity and showed a restricted biodistribution to the lesion. Neuropathological examination of the CNS revealed no adverse effects of HuSCs. Animals exhibiting higher numbers of surviving HuSCs within the lesion showed greater volumes of preserved white matter and host rat SC and astrocyte ingress as well as axon ingrowth and myelination. These results demonstrate the safety of HuSCs when employed in a clinically relevant experimental SCI paradigm. Further, signs of a potentially positive influence of HuSC transplants on host tissue pathology were observed. These findings show that HuSCs exhibit a favorable toxicity profile for up to 6 months after transplantation into the contused rat spinal cord, an important outcome for FDA consideration of their use in human clinical trials.

Defective synthesis and release of astrocytic thrombospondin-1 mediates the neuronal TDP-43 proteinopathy, resulting in defects in neuronal integrity associated with chronic traumatic encephalopathy: in vitro studies.

Transactivating DNA-binding protein-43 (TDP-43) inclusions and the accumulation of phosphorylated and ubiquitinated tau proteins (p-tau) have been identified in postmortem brain specimens from patients with chronic traumatic encephalopathy (CTE). To examine whether these proteins contribute to the development of CTE, we utilized an in vitro trauma system known to reproduce many of the findings observed in humans and experimental animals with traumatic brain injury. Accordingly, we examined the role of TDP-43 and Tau in an in vitro model of trauma, and determined whether these proteins contribute to the defective neuronal integrity associated with CNS trauma. Single or multiple episodes of trauma to cultured neurons resulted in a time-dependent increase in cytosolic levels of phosphorylated TDP-43 (p-TDP-43). Trauma to cultured neurons also caused an increase in levels of casein kinase 1 epsilon (CK1ε), and ubiquitinated p-TDP-43, along with a decrease in importin-ß (all factors known to mediate the "TDP-43 proteinopathy"). Defective neuronal integrity, as evidenced by a reduction in levels of the NR1 subunit of the NMDA receptor, and in PSD95, along with increased levels of phosphorylated tau were also observed. Additionally, increased levels of intra- and extracellular thrombospondin-1 (TSP-1) (a factor known to regulate neuronal integrity) were observed in cultured astrocytes at early stages of trauma, while at later stages decreased levels were identified. The addition of recombinant TSP-1, conditioned media from cultured astrocytes at early stages of trauma, or the CK1ε inhibitor PF4800567 hydrochloride to traumatized cultured neurons reduced levels of p-TDP-43, and reversed the trauma-induced decline in NR1 subunit of the NMDA receptor and PSD95 levels. These findings suggest that a trauma-induced increase in TDP-43 phosphorylation contributes to defective neuronal integrity, and that increasing TSP-1 levels may represent a useful therapeutic approach for the prevention of the neuronal TDP-43 proteinopathy associated with CTE. Read the Editorial Highlight for this article on page 531.

Decreased STAT3 Phosphorylation Mediates Cell Swelling in Ammonia-Treated Astrocyte Cultures.

Brain edema, due largely to astrocyte swelling, and the subsequent increase in intracranial pressure and brain herniation, are major complications of acute liver failure (ALF). Elevated level of brain ammonia has been strongly implicated in the development of astrocyte swelling associated with ALF. The means by which ammonia brings about astrocyte swelling, however, is incompletely understood. Recently, oxidative/nitrosative stress and associated signaling events, including activation of mitogen-activated protein kinases (MAPKs), as well as activation of the transcription factor, nuclear factor-kappaB (NF-κB), have been implicated in the mechanism of ammonia-induced astrocyte swelling. Since these signaling events are known to be regulated by the transcription factor, signal transducer and activator of transcription 3 (STAT3), we examined the state of STAT3 activation in ammonia-treated cultured astrocytes, and determined whether altered STAT3 activation and/or protein expression contribute to the ammonia-induced astrocyte swelling. STAT3 was found to be dephosphorylated (inactivated) at Tyrosine705 in ammonia-treated cultured astrocytes. Total STAT3 protein level was also reduced in ammonia-treated astrocytes. We also found a significant increase in protein tyrosine phosphatase receptor type-1 (PTPRT-1) protein expression in ammonia-treated cultured astrocytes, and that inhibition of PTPRT-1 enhanced the phosphorylation of STAT3 after ammonia treatment. Additionally, exposure of cultured astrocytes to inhibitors of protein tyrosine phosphatases diminished the ammonia-induced cell swelling, while cultured astrocytes over-expressing STAT3 showed a reduction in the astrocyte swelling induced by ammonia. Collectively, these studies strongly suggest that inactivation of STAT3 represents a critical event in the mechanism of the astrocyte swelling associated with acute liver failure.

Glutamine Synthetase: Role in Neurological Disorders.

Glutamine synthetase (GS) is an ATP-dependent enzyme found in most species that synthesizes glutamine from glutamate and ammonia. In brain, GS is exclusively located in astrocytes where it serves to maintain the glutamate-glutamine cycle, as well as nitrogen metabolism. Changes in the activity of GS, as well as its gene expression, along with excitotoxicity, have been identified in a number of neurological conditions. The literature describing alterations in the activation and gene expression of GS, as well as its involvement in different neurological disorders, however, is incomplete. This review summarizes changes in GS gene expression/activity and its potential contribution to the pathogenesis of several neurological disorders, including hepatic encephalopathy, ischemia, epilepsy, Alzheimer's disease, amyotrophic lateral sclerosis, traumatic brain injury, Parkinson's disease, and astroglial neoplasms. This review also explores the possibility of targeting GS in the therapy of these conditions.

Neuroinflammation in hepatic encephalopathy: mechanistic aspects.

Hepatic encephalopathy (HE) is a major neurological complication of severe liver disease that presents in acute and chronic forms. While elevated brain ammonia level is known to be a major etiological factor in this disorder, recent studies have shown a significant role of neuroinflammation in the pathogenesis of both acute and chronic HE. This review summarizes the involvement of ammonia in the activation of microglia, as well as the means by which ammonia triggers inflammatory responses in these cells. Additionally, the role of ammonia in stimulating inflammatory events in brain endothelial cells (ECs), likely through the activation of the toll-like receptor-4 and the associated production of cytokines, as well as the stimulation of various inflammatory factors in ECs and in astrocytes, are discussed. This review also summarizes the inflammatory mechanisms by which activation of ECs and microglia impact on astrocytes leading to their dysfunction, ultimately contributing to astrocyte swelling/brain edema in acute HE. The role of microglial activation and its contribution to the progression of neurobehavioral abnormalities in chronic HE are also briefly presented. We posit that a better understanding of the inflammatory events associated with acute and chronic HE will uncover novel therapeutic targets useful in the treatment of patients afflicted with HE.

Expression of glutamine transporter isoforms in cerebral cortex of rats with chronic hepatic encephalopathy.

Hepatic encephalopathy (HE) is a neuropsychiatric disorder that occurs due to acute and chronic liver diseases, the hallmark of which is the increased levels of ammonia and subsequent alterations in glutamine synthesis, i.e. conditions associated with the pathophysiology of HE. Under physiological conditions, glutamine is fundamental for replenishment of the neurotransmitter pools of glutamate and GABA. The different isoforms of glutamine transporters play an important role in the transfer of this amino acid between astrocytes and neurons. A disturbance in the GABA biosynthetic pathways has been described in bile duct ligated (BDL) rats, a well characterized model of chronic HE. Considering that glutamine is important for GABA biosynthesis, altered glutamine transport and the subsequent glutamate/GABA-glutamine cycle efficacy might influence these pathways. Given this potential outcome, the aim of the present study was to investigate whether the expression of the glutamine transporters SAT1, SAT2, SN1 and SN2 would be affected in chronic HE. We verified that mRNA expression of the neuronal glutamine transporters SAT1 and SAT2 was found unaltered in the cerebral cortex of BDL rats. Similarly, no changes were found in the mRNA level for the astrocytic transporter SN1, whereas the gene expression of SN2 was increased by two-fold in animals with chronic HE. However, SN2 protein immuno-reactivity did not correspond with the increase in gene transcription since it remained unaltered. These data indicate that the expression of the glutamine transporter isoforms is unchanged during chronic HE, and thus likely not to participate in the pathological mechanisms related to the imbalance in the GABAergic neurotransmitter system observed in this neurologic condition.

Axonal regeneration. Systemic administration of epothilone B promotes axon regeneration after spinal cord injury.

After central nervous system (CNS) injury, inhibitory factors in the lesion scar and poor axon growth potential prevent axon regeneration. Microtubule stabilization reduces scarring and promotes axon growth. However, the cellular mechanisms of this dual effect remain unclear. Here, delayed systemic administration of a blood-brain barrier-permeable microtubule-stabilizing drug, epothilone B (epoB), decreased scarring after rodent spinal cord injury (SCI) by abrogating polarization and directed migration of scar-forming fibroblasts. Conversely, epothilone B reactivated neuronal polarization by inducing concerted microtubule polymerization into the axon tip, which propelled axon growth through an inhibitory environment. Together, these drug-elicited effects promoted axon regeneration and improved motor function after SCI. With recent clinical approval, epothilones hold promise for clinical use after CNS injury.

Expression of Gls and Gls2 glutaminase isoforms in astrocytes.

The expression of glutaminase in glial cells has been a controversial issue and matter of debate for many years. Actually, glutaminase is essentially considered as a neuronal marker in brain. Astrocytes are endowed with efficient and high capacity transport systems to recapture synaptic glutamate which seems to be consistent with the absence of glutaminase in these glial cells. In this work, a comprehensive study was devised to elucidate expression of glutaminase in neuroglia and, more concretely, in astrocytes. Immunocytochemistry in rat and human brain tissues employing isoform-specific antibodies revealed expression of both Gls and Gls2 glutaminase isozymes in glutamatergic and GABAergic neuronal populations as well as in astrocytes. Nevertheless, there was a different subcellular distribution: Gls isoform was always present in mitochondria while Gls2 appeared in two different locations, mitochondria and nucleus. Confocal microscopy and double immunofluorescence labeling in cultured astrocytes confirmed the same pattern previously seen in brain tissue samples. Astrocytic glutaminase expression was also assessed at the mRNA level, real-time quantitative RT-PCR detected transcripts of four glutaminase isozymes but with marked differences on their absolute copy number: the predominance of Gls isoforms over Gls2 transcripts was remarkable (ratio of 144:1). Finally, we proved that astrocytic glutaminase proteins possess enzymatic activity by in situ activity staining: concrete populations of astrocytes were labeled in the cortex, cerebellum and hippocampus of rat brain demonstrating functional catalytic activity. These results are relevant for the stoichiometry of the Glu/Gln cycle at the tripartite synapse and suggest novel functions for these classical metabolic enzymes.

Decreased astrocytic thrombospondin-1 secretion after chronic ammonia treatment reduces the level of synaptic proteins: in vitro and in vivo studies.

Chronic hepatic encephalopathy (CHE) is a major complication in patients with severe liver disease. Elevated blood and brain ammonia levels have been implicated in its pathogenesis, and astrocytes are the principal neural cells involved in this disorder. Since defective synthesis and release of astrocytic factors have been shown to impair synaptic integrity in other neurological conditions, we examined whether thrombospondin-1 (TSP-1), an astrocytic factor involved in the maintenance of synaptic integrity, is also altered in CHE. Cultured astrocytes were exposed to ammonia (NH4Cl, 0.5-2.5 mM) for 1-10 days, and TSP-1 content was measured in cell extracts and culture media. Astrocytes exposed to ammonia exhibited a reduction in intra- and extracellular TSP-1 levels. Exposure of cultured neurons to conditioned media from ammonia-treated astrocytes showed a decrease in synaptophysin, PSD95, and synaptotagmin levels. Conditioned media from TSP-1 over-expressing astrocytes that were treated with ammonia, when added to cultured neurons, reversed the decline in synaptic proteins. Recombinant TSP-1 similarly reversed the decrease in synaptic proteins. Metformin, an agent known to increase TSP-1 synthesis in other cell types, also reversed the ammonia-induced TSP-1 reduction. Likewise, we found a significant decline in TSP-1 level in cortical astrocytes, as well as a reduction in synaptophysin content in vivo in a rat model of CHE. These findings suggest that TSP-1 may represent an important therapeutic target for CHE. Defective release of astrocytic factors may impair synaptic integrity in chronic hepatic encephalopathy. We found a reduction in the release of the astrocytic matricellular proteins thrombospondin-1 (TSP-1) in ammonia-treated astrocytes; such reduction was associated with a decrease in synaptic proteins caused by conditioned media from ammonia-treated astrocytes. Exposure of neurons to CM from ammonia-treated astrocytes, in which TSP-1 is over-expressed, reversed (by approx 75%) the reduction in synaptic proteins. NF-kB = nuclear factor kappa B; PSD95 = post-synaptic density protein 95; ONS = oxidative/nitrative stress.