Response of Astrocyte Subpopulations Following Spinal Cord Injury.

There is growing appreciation for astrocyte heterogeneity both across and within central nervous system (CNS) regions, as well as between intact and diseased states. Recent work identified multiple astrocyte subpopulations in mature brain. Interestingly, one subpopulation (Population C) was shown to possess significantly enhanced synaptogenic properties in vitro, as compared with other astrocyte subpopulations of adult cortex and spinal cord. Following spinal cord injury (SCI), damaged neurons lose synaptic connections with neuronal partners, resulting in persistent functional loss. We determined whether SCI induces an enhanced synaptomodulatory astrocyte phenotype by shifting toward a greater proportion of Population C cells and/or increasing expression of relevant synapse formation-associated genes within one or more astrocyte subpopulations. Using flow cytometry and RNAscope in situ hybridization, we found that astrocyte subpopulation distribution in the spinal cord did not change to a selectively synaptogenic phenotype following mouse cervical hemisection-type SCI. We also found that spinal cord astrocytes expressed synapse formation-associated genes to a similar degree across subpopulations, as well as in an unchanged manner between uninjured and SCI conditions. Finally, we confirmed these astrocyte subpopulations are also present in the human spinal cord in a similar distribution as mouse, suggesting possible conservation of spinal cord astrocyte heterogeneity across species.

Facial grimace testing as an assay of neuropathic pain-related behavior in a mouse model of cervical spinal cord injury.

A major portion of individuals affected by traumatic spinal cord injury (SCI) experience one or more types of chronic neuropathic pain (NP), which is often intractable to currently available treatments. The availability of reliable behavioral assays in pre-clinical models of SCI-induced NP is therefore critical to assess the efficacy of new potential therapies. Commonly used assays to evaluate NP-related behavior in rodents, such as Hargreaves thermal and von Frey mechanical testing, rely on the withdrawal response to an evoked stimulus. However, other assays that test spontaneous/non-evoked NP-related behavior or supraspinal aspects of NP would be highly useful for a more comprehensive assessment of NP following SCI. The Mouse Grimace Scale (MGS) is a tool to assess spontaneous, supraspinal pain-like behaviors in mice; however, the assay has not been characterized in a mouse model of SCI-induced chronic NP, despite the critical importance of mouse genetics as an experimental tool. We found that beginning 2 weeks after cervical contusion, SCI mice exhibited increased facial grimace features compared to laminectomy-only control mice, and this grimace phenotype persisted to the chronic time point of 5 weeks post-injury. We also found a significant relationship between facial grimace score and the evoked forepaw withdrawal response in both the Hargreaves and von Frey tests at 5 weeks post-injury when both laminectomy-only and SCI mice were included in the analysis. However, within only the SCI group, there was no correlation between grimace score and Hargreaves or von Frey responses. These results indicate both that facial grimace analysis can be used as an assay of spontaneous NP-related behavior in the mouse model of SCI and that the information provided by the MGS may be different than that provided by evoked tests of sensory function.

Sonic hedgehog signaling is negatively regulated in reactive astrocytes after forebrain stab injury.

Following injury to the central nervous system, astrocytes perform critical and complex functions that both promote and antagonize neural repair. Understanding the molecular signaling pathways that coordinate their diverse functional properties is key to developing effective therapeutic strategies. In the healthy, adult CNS, Sonic hedgehog (Shh) signaling is active in mature, differentiated astrocytes. Shh has been shown to undergo injury-induced upregulation and promote neural repair. Here, we investigated whether Shh signaling mediates astrocyte response to injury. Surprisingly, we found that following an acute, focal injury, reactive astrocytes exhibit a pronounced reduction in Shh activity in a spatiotemporally-defined manner. Shh signaling is lost in reactive astrocytes at the lesion site, but persists in mild to moderately reactive astrocytes in distal tissues. Nevertheless, local pharmacological activation of the Shh pathway in astrocytes mitigates inflammation, consistent with a neuroprotective role for Shh signaling after injury. Interestingly, we find that Shh signaling is restored to baseline levels two weeks after injury, a time during which acute inflammation has largely subsided and lesions have matured. Taken together, these data suggest that endogenous Shh signaling in astrocytes is dynamically regulated in a context dependent manner. In addition, exogenous activation of the Shh pathway promotes neuroprotection mediated by reactive astrocytes.

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury.

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive astrogliosis. Reactive astrocytes exert both inflammatory and protective effects that inhibit and promote, respectively, neural repair. The mechanisms underlying the diverse functional properties of reactive astrogliosis are not well understood. Achieving a greater understanding of these mechanisms is critical to developing therapeutic strategies to treat the injured CNS. Here we demonstrate a method to trigger reactive astrogliosis in the adult mouse forebrain using a forebrain stab lesion. This lesion model is simple, reliable, and requires only a stereotaxic device and a scalpel blade to produce the injury. The use of stab lesions as an injury model in the forebrain is well established and amenable to studies addressing a broad range of neuropathological outcomes, such as neuronal degeneration, neuroinflammation, and disruptions in the blood brain barrier (BBB). Thus, the forebrain stab injury model serves as a powerful tool that can be applied for a broad range of studies on the CNS response to trauma.

Effects of an NADPH-generating system on primaquine degradation by hamster liver fractions.

1. Primaquine (PQ) often causes severe anaemia in individuals with glucose 6-phosphate dehydrogenase (G6PD) deficient erythrocytes, and metabolites have been implicated as the toxic substance. These studies present data identifying additional metabolites of PQ. 2. Two metabolites of primaquine (PQ) previously identified in human studies, namely, 6-methoxy-8-aminoquinoline (MAQ) and 8-(3-carboxy-1-methylpropylamino)-6-methoxyquinoline (PQC) were also formed on incubation of PQ with hamster liver fractions for up to 24 h without an NADPH-generating system. 3. The alcohol (PQAOH) and lactam (PQLT) derivatives of PQ were also formed on incubation with hamster liver fraction used in these studies. 4. The microsomal metabolism of PQ was decreased in presence of an NADPH-generating system, but not by SKF-525A or glutathione (GSH) indicating that the oxidative reactions were probably not due to the cytochrome P-450 system or free radical mechanisms.

Ellagic acid metabolism and binding to DNA in organ explant cultures of the rat.

Ellagic acid (EA) is a plant phenolic compound with postulated antimutagenic and anticarcinogenic activity. In this study, explants of esophagus, forestomach, colon, bladder, trachea, lung and liver from male Sprague-Dawley rats (130-140 g) were incubated in culture medium containing [3H]EA (20 microM, 4.5 microCi/ml) for 24 h at 37 degrees C. After extraction, purification and quantitation of explant DNA significant differences in the binding of EA to the DNA was observed. The most binding occurred in esophagus and the least in lung. Analysis of the organsoluble fraction of the culture medium by high performance liquid chromatography yielded 3 metabolites of EA. None of the metabolites were identified. Elution of water-soluble metabolites from an alumina column showed that there were sulfate ester, glucuronide and glutathione conjugates of EA in the explant culture medium from all the organs. The profile of water-soluble conjugates was very similar between colon and forestomach and between trachea and lung. These results indicate that EA binds to DNA in different tissues and that tissues metabolize EA to both organosoluble and water-soluble products.

Synthesis of certain hydroxy analogues of the antimalarial drug primaquine and their in vitro methemoglobin-producing and glutathione-depleting activity in human erythrocytes.

A number of hydroxy analogues of the antimalarial drug primaquine [8-[(4-amino-1-methylbutyl)amino]-6-methoxyquinoline] were synthesized and characterized by 1H NMR and mass spectra. Several of the compounds were found to be active in forming methemoglobin in human erythrocytes, particularly in those from glucose-6-phosphate dehydrogenase (G6PD) deficient subjects. Decreased levels of glutathione (GSH) in G6PD-deficient erythrocytes were also found with compounds that were active methemoglobin formers.

In vitro metabolism of the antimalarial agent primaquine by mouse liver enzymes and identification of a methemoglobin-forming metabolite.

From a mouse liver microsomal system, we have isolated and identified a methemoglobin-forming metabolite of primaquine (PQ). Evidence has been found for both O-dealkylation and hydroxylation of PQ to form a metabolite, 5,6-dihydroxy-8-(4-amino-1-methylbutylamino)quinoline, which is highly active in forming methemoglobin in both normal and glucose-6-phosphate dehydrogenase-deficient erythrocytes. It also actively decreases glutathione levels in glucose-6-phosphate dehydrogenase-deficient erythrocytes. The inhibitor SKF 525-A prevented metabolite formation while iproniazid and carbon monoxide did not inhibit metabolism completely but may have resulted in formation of a different unidentified metabolite. Mass spectrometry, HPLC, NMR, and other more indirect methods were used to help identify the metabolite. It was identified indirectly via a blue compound which results from extracting the actual metabolite from the incubation mixture with organic solvents under alkaline conditions in the presence of light. The blue compound was identified as a quinonimine in which the 8-amino side chain of PQ cyclizes to produce a third ring system.

Metabolism of 8-aminoquinoline antimalarial agents.

Some of the most effective antimalarial agents are derivatives of 8-aminoquinoline. The metabolic products of many of these compounds appear to be toxic to the erythrocytes of certain human subjects, especially those deficient in glucose-6-phosphate dehydrogenase. Although a number of studies have been conducted over many years, the metabolism of most of these compounds has not been determined. These studies are reviewed.Adult dogs dosed with tritium-labelled primaquine were observed to excrete approximately 16% of the injected radioactivity in the urine within 8 hours. Organic extracts of the urine were fractionated by thin-layer chromatography and the metabolic pattern obtained. Some primaquine was excreted along with at least five metabolites including 5-hydroxy-6-methoxy-8-(4-amino-1-methylbutylamino)quinoline (5HPQ) and a small amount of 6-hydroxy-8-(4-amino-1-methylbutylamino)quinoline (6HPQ). The 5HPQ could form a quinoneimine-type compound which may be a methaemoglobin-forming compound. This and other metabolites isolated from urine were found to be active methaemoglobin formers in in vitro studies. In vitro metabolism of primaquine by mouse liver enzymes also produced compounds capable of methaemoglobin formation. One of these had a blue colour when exposed to alkaline conditions, air, and light, and mass spectral data and nuclear magnetic resonance analysis indicated a structure similar to a 5,6-dihydroxy derivative of primaquine. However, the chemical structure of the metabolite was not identified in these studies.

Absorption and metabolism of the chiral isomers of fonofos in the corn and cotton plant.

Root absorption of chiral phenyl-35S-fonofos in cotton and corn plants revealed stereoselective differences between the two enantiomers. (S)p-Fonofos was absorbed at a faster initial rate and to a greater extent than the (R)p enantiomer in both plant species. Approximately 40% and 62% of the applied radioactivity was absorbed into the cotton plant 12 hr after application of (R)p- and (S)p-fonofos, respectively. In the corn plant, approximately 25% and 63% of the applied (R)p- and (S)p-fonofos was absorbed in the first 12 hrs. Little qualitative or quantitative difference in plant translocation between fonofos enantiomers was observed. (R)p-fonofos was found to be metabolized to a greater extent than the (S)p enantiomer in both cotton and corn plants.