l-α-aminoadipate causes astrocyte pathology with negative impact on mouse hippocampal synaptic plasticity and memory.

Increasing evidence shows that astrocytes, by releasing and uptaking neuroactive molecules, regulate synaptic plasticity, considered the neurophysiological basis of memory. This study investigated the impact of l-α-aminoadipate (l-AA) on astrocytes which sense and respond to stimuli at the synaptic level and modulate hippocampal long-term potentiation (LTP) and memory. l-AA selectivity toward astrocytes was proposed in the early 70's and further tested in different systems. Although it has been used for impairing the astrocytic function, its effects appear to be variable in different brain regions. To test the effects of l-AA in the hippocampus of male C57Bl/6 mice we performed two different treatments (ex vivo and in vivo) and took advantage of other compounds that were reported to affect astrocytes. l-AA superfusion did not affect the basal synaptic transmission but decreased LTP magnitude. Likewise, trifluoroacetate and dihydrokainate decreased LTP magnitude and occluded the effect of l-AA on synaptic plasticity, confirming l-AA selectivity. l-AA superfusion altered astrocyte morphology, increasing the length and complexity of their processes. In vivo, l-AA intracerebroventricular injection not only reduced the astrocytic markers but also LTP magnitude and impaired hippocampal-dependent memory in mice. Interestingly, d-serine administration recovered hippocampal LTP reduction triggered by l-AA (2 h exposure in hippocampal slices), whereas in mice injected with l-AA, the superfusion of d-serine did not fully rescue LTP magnitude. Overall, these data show that both l-AA treatments affect astrocytes differently, astrocytic activation or loss, with similar negative outcomes on hippocampal LTP, implying that opposite astrocytic adaptive alterations are equally detrimental for synaptic plasticity.

Hippocampus-based contextual memory alters the morphological characteristics of astrocytes in the dentate gyrus.

Astrocytes have been reported to exist in two states, the resting and the reactive states. Morphological changes in the reactive state of astrocytes include an increase in thickness and number of processes, and an increase in the size of the cell body. Molecular changes also occur, such as an increase in the expression of glial fibrillary acidic protein (GFAP). However, the morphological and molecular changes during the process of learning and memory have not been elucidated. In the current study, we subjected Fvb/n mice to contextual fear conditioning, and checked for morphological and molecular changes in astrocytes. 1 h after fear conditioning, type II and type III astrocytes exhibited a unique status with an increased number of processes and decreased GFAP expression which differed from the typical resting or reactive state. In addition, the protein level of excitatory excitatory amino acid transporter 2 (EAAT2) was increased 1 h to 24 h after contextual fear conditioning while EAAT1 did not show any alterations. Connexin 43 (Cx43) protein was found to be increased at 24 h after fear conditioning. These data suggest that hippocampus-based contextual memory process induces changes in the status of astrocytes towards a novel status different from typical resting or reactive states. These morphological and molecular changes may be in line with functional changes.

Reshaping the Cone-Mosaic in a Rat Model of Retinitis Pigmentosa: Modulatory Role of ZO-1 Expression in DL-Alpha-Aminoadipic Acid Reshaping.

In S334ter-line-3 rat model of Retinitis Pigmentosa (RP), rod cell death induces the rearrangement of cones into mosaics of rings while the fibrotic processes of Müller cells remodel to fill the center of the rings. In contrast, previous work established that DL-alpha-aminoadipic-acid (AAA), a compound that transiently blocks Müller cell metabolism, abolishes these highly structured cone rings. Simultaneously, adherens-junction associated protein, Zonula occludens-1 (ZO-1) expression forms in a network between the photoreceptor segments and Müller cells processes. Thus, we hypothesized that AAA treatment alters the cone mosaic rings by disrupting the distal sealing formed by these fibrotic processes, either directly or indirectly, by down regulating the expression of ZO-1. Therefore, we examined these processes and ZO-1 expression at the outer retina after intravitreal injection of AAA and observed that AAA treatment transiently disrupts the distal glial sealing in RP retina, plus induces cones in rings to become more homogeneous. Moreover, ZO-1 expression is actively suppressed after 3 days of AAA treatment, which coincided with cone ring disruption. Similar modifications of glial sealing and cone distribution were observed after injection of siRNA to inhibit ZO-1 expression. These findings support our hypothesis and provide additional information about the critical role played by ZO-1 in glial sealing and shaping the ring mosaic in RP retina. These studies represent important advancements in the understanding of retinal degeneration's etiology and pathophysiology.

Activated spinal astrocytes are involved in the maintenance of chronic widespread mechanical hyperalgesia after cast immobilization.

BACKGROUND: In the present study, we examined spinal glial cell activation as a central nervous system mechanism of widespread mechanical hyperalgesia in rats that experienced chronic post-cast pain (CPCP) 2 weeks after cast immobilization. Activated spinal microglia and astrocytes were investigated immunohistologically in lumbar and coccygeal spinal cord segments 1 day, 5 weeks, and 13 weeks following cast removal. RESULTS: In the lumbar cord, astrocytes were activated after microglia. Astrocytes also were activated after microglia in the coccygeal cord, but with a delay that was longer than that observed in the lumbar cord. This activation pattern paralleled the observation that mechanical hyperalgesia occurred in the hindleg or the hindpaw before the tail. The activating transcription factor 3 (ATF3) immune response in dorsal root ganglia (DRG) on the last day of cast immobilization suggested that nerve damage might not occur in CPCP rats. The neural activation assessed by the phosphorylated extracellular signal-regulated kinase (pERK) immune response in DRG arose 1 day after cast removal. In addition, L-α-aminoadipate (L-α-AA), an inhibitor of astrocyte activation administered intrathecally 5 weeks after cast removal, inhibited mechanical hyperalgesia in several body parts including the lower leg skin and muscles bilaterally, hindpaws, and tail. CONCLUSIONS: These findings suggest that activation of lumbar cord astrocytes is an important factor in widespread mechanical hyperalgesia in CPCP.

Astrocyte pathology in the prefrontal cortex impairs the cognitive function of rats.

Interest in astroglial cells is rising due to recent findings supporting dynamic neuron-astrocyte interactions. There is increasing evidence of astrocytic dysfunction in several brain disorders such as depression, schizophrenia or bipolar disorder; importantly these pathologies are characterized by the involvement of the prefrontal cortex and by significant cognitive impairments. Here, to model astrocyte pathology, we injected animals with the astrocyte specific toxin L-α-aminoadipate (L-AA) in the medial prefrontal cortex (mPFC); a behavioral and structural characterization two and six days after the injection was performed. Behavioral data shows that the astrocyte pathology in the mPFC affects the attentional set-shifting, the working memory and the reversal learning functions. Histological analysis of brain sections of the L-AA-injected animals revealed a pronounced loss of astrocytes in the targeted region. Interestingly, analysis of neurons in the lesion sites showed a progressive neuronal loss that was accompanied with dendritic atrophy in the surviving neurons. These results suggest that the L-AA-induced astrocytic loss in the mPFC triggers subsequent neuronal damage leading to cognitive impairment in tasks depending on the integrity of this brain region. These findings are of relevance to better understand the pathophysiological mechanisms underlying disorders that involve astrocytic loss/dysfunction in the PFC.

Role of Müller cells in cone mosaic rearrangement in a rat model of retinitis pigmentosa.

Retinitis pigmentosa (RP) is a type of inherited retinal degenerative disease, which leads to blindness. The primary pathological event of this disease is the death of rods because of genetic mutations. The S334ter-line-3 rat is a transgenic model developed to express a rhodopsin mutation similar to that found in RP. In this study, the rod's death triggered are organization of the cone mosaic into an orderly array of rings. Four observations were relevant to understand this reorganization. First, rods died in hot spots, which progressively increased as circular waves, leaving rod-less zones behind. Second, rings of cones formed around these zones. Third, remodeled Müller glia processes enveloped cones and filled the center of their rings. Zonula occludens-1 located between the photoreceptor inner segments and the apical processes of Müller cells showed in the rings. Fourth, these rings were formed before the onset of cone cell deaths and were maintained until late stages of RP. From these observations,we hypothesize that cone-Müller-cell interactions mediate and maintain the rings. A test of this hypothesis can be performed by injecting DL-a-aminoadipic acid (AAA), which is known to disrupt Müller cell metabolism. A single intravitreal injection of AAA at P50 disrupted the rings of cones 3 days after the injection. These findings indicate that the rearrangement of cones in rings is modulated by Müller cells in RP. Thus, if the relationship between photoreceptors and Müller glia is better understood, the latter could potentially be manipulated for effective neuroprotection or the restoration of normal cone arrays.

Spinal astrocytic activation contributes to mechanical allodynia in a mouse model of type 2 diabetes.

Diabetic neuropathic pain (DNP) plays a major role in decreased life quality of type 2 diabetes patients, however, the molecular mechanisms underlying DNP remain unclear. Emerging research implicates the participation of spinal glial cells in some neuropathic pain models. However, it remains unknown whether spinal glial cells are activated under type 2 diabetic conditions and whether they contribute to diabetes-induced neuropathic pain. In the present study, using a db/db type 2 diabetes mouse model that displayed obvious mechanical allodynia, we found that spinal astrocyte but not microglia was dramatically activated. The mechanical allodynia was significantly attenuated by intrathecally administrated l-α-aminoadipate (astrocytic specific inhibitor) whereas minocycline (microglial specific inhibitor) did not have any effect on mechanical allodynia, which indicated that spinal astrocytic activation contributed to allodynia in db/db mice. Further study aimed to identify the detailed mechanism of astrocyte-induced allodynia in db/db mice. Results showed that spinal activated astrocytes dramatically increased interleukin (IL)-1ß expression which may induce N-methyl-D-aspartic acid receptor (NMDAR) phosphorylation in spinal dorsal horn neurons to enhance pain transmission. Together, these results suggest that spinal activated astrocytes may be a crucial component of mechanical allodynia in type 2 diabetes and "Astrocyte-IL-1ß-NMDAR-Neuron" pathway may be the detailed mechanism of astrocyte-induced allodynia. Thus, inhibiting astrocytic activation in the spinal dorsal horn may represent a novel therapeutic strategy for treating DNP.

Combining ketamine with astrocytic inhibitor as a potential analgesic strategy for neuropathic pain ketamine, astrocytic inhibitor and pain.

BACKGROUND: Neuropathic pain is an intractable clinical problem. Intrathecal ketamine, a noncompetitive N-methyl-D-aspartate receptor (NMDAR) antagonist, is reported to be useful for treating neuropathic pain in clinic by inhibiting the activity of spinal neurons. Nevertheless, emerging studies have disclosed that spinal astrocytes played a critical role in the initiation and maintenance of neuropathic pain. However, the present clinical therapeutics is still just concerning about neuronal participation. Therefore, the present study is to validate the coadministration effects of a neuronal noncompetitive N-methyl-D-aspartate receptor (NMDAR) antagonist ketamine and astrocytic cytotoxin L-α-aminoadipate (LAA) on spinal nerve ligation (SNL)-induced neuropathic pain. RESULTS: Intrathecal ketamine (10, 100, 1000 µg/kg) or LAA (10, 50, 100 nmol) alleviated SNL-induced mechanical allodynia in a dose-dependent manner respectively. Phosphorylated NR1 (pNR1) or glial fibrillary acidic protein (GFAP) expression was down-regulated by intrathecal ketamine (100, 1000 µg/kg) or LAA (50, 100 nmol) respectively. The combination of ketamine (100 µg/kg) with LAA (50 nmol) showed superadditive effects on neuropathic pain compared with that of intrathecal administration of either ketamine or LAA alone. Combined administration obviously relieved mechanical allodynia in a quick and stable manner. Moreover, down-regulation of pNR1 and GFAP expression were also enhanced by drugs coadministration. CONCLUSIONS: These results suggest that combining NMDAR antagonist ketamine with an astrocytic inhibitor or cytotoxin, which is suitable for clinical use once synthesized, might be a potential strategy for clinical management of neuropathic pain.

Pharmacological disruption of the outer limiting membrane leads to increased retinal integration of transplanted photoreceptor precursors.

Retinal degeneration is the leading cause of untreatable blindness in the developed world. Cell transplantation strategies provide a novel therapeutic approach to repair the retina and restore sight. Previously, we have shown that photoreceptor precursor cells can integrate and form functional photoreceptors after transplantation into the subretinal space of the adult mouse. In a clinical setting, however, it is likely that far greater numbers of integrated photoreceptors would be required to restore visual function. We therefore sought to assess whether the outer limiting membrane (OLM), a natural barrier between the subretinal space and the outer nuclear layer (ONL), could be reversibly disrupted and if disruption of this barrier could lead to enhanced numbers of transplanted photoreceptors integrating into the ONL. Transient chemical disruption of the OLM was induced in adult mice using the glial toxin, dl-alpha-aminoadipic acid (AAA). Dissociated early post-natal neural retinal cells were transplanted via subretinal injection at various time-points after AAA administration. At 3 weeks post-injection, the number of integrated, differentiated photoreceptor cells was assessed and compared with those found in the PBS-treated contralateral eye. We demonstrate for the first time that the OLM can be reversibly disrupted in adult mice, using a specific dose of AAA administered by intravitreal injection. In this model, OLM disruption is maximal at 72 h, and recovers by 2 weeks. When combined with cell transplantation, disruption of the OLM leads to a significant increase in the number of photoreceptors integrated within the ONL compared with PBS-treated controls. This effect was only seen in animals in which AAA had been administered 72 h prior to transplantation, i.e. when precursor cells were delivered into the subretinal space at a time coincident with maximal OLM disruption. These findings suggest that the OLM presents a physical barrier to photoreceptor integration following transplantation into the subretinal space in the adult mouse. Reversible disruption of the OLM may provide a strategy for increasing cell integration in future therapeutic applications.

Effect of MX-68 on airway inflammation and hyperresponsiveness in mice and guinea-pigs.

MX-68 is a newly synthesized antifolate, which is a derivative of methotrexate (MTX). In this paper, the effect of MX-68 on allergic airway responses in mice and guinea-pigs was studied. In the first experiment, antigen-induced airway inflammation and airway hyperresponsiveness (AHR) to acetylcholine in mice were examined and compared with the effects of classical antifolate methotrexate and prednisolone. Mice were sensitized with ovalbumin as an antigen and challenged with ovalbumin inhalation three times. After the last inhalation, AHR and airway inflammation were observed. An increase in Th2 cytokines (IL-4 and IL-5) and a decrease in a Th1 cytokine (IFN-gamma) in the bronchoalveolar lavage fluid (BALF), as well as an elevation of the immunoglobulin level in serum, were observed in sensitized mice. Oral administration of MX-68 had no effect on changes of body weight, but prednisolone reduced body weight during the experiment. The antigen-induced AHR and increases in the number of eosinophils and lymphocytes in BALF were significantly inhibited by MX-68. MX-68 interfered with the elevation of IL-4 and IL-5 levels in BALF, but had no effect on the decrease in IFN-gamma. Moreover, MX-68 significantly inhibited the elevation of serum IgE and IgG levels. In the guinea-pig model for bronchial asthma, biphasic increases in airway resistance (immediate asthmatic response, IAR, and late asthmatic response, LAR), as well as accumulated inflammatory cells in BALF, were observed after repeated antigen challenge. These asthmatic responses and inflammatory signs were significantly decreased by administration of MX-68. These results suggest that MX-68 obviously has an anti-inflammatory effect in an animal model of asthma and would be useful clinically for the treatment of bronchial asthma.

Twenty-four-hour intravenous and oral tracer studies with L-[1-13C]-2-aminoadipic acid and L-[1-13C]lysine as tracers at generous nitrogen and lysine intakes in healthy adults.

BACKGROUND: This is a continuation of investigations of the relations between amino acid kinetics and amino acid dietary requirements in healthy adults. OBJECTIVE: The aim was to investigate the 24-h pattern and rate of the metabolism of an L-[1-13C]-2-aminoadipic acid ([13C]AAA) tracer and of whole-body L-[1-13C]lysine ([13C]lysine) oxidation and balance in healthy, young adults receiving a generous intake of lysine. DESIGN: Thirteen healthy adults were given an adequate, L-amino acid-based diet supplying 77 mg lysine x kg(-1) x d(-1) for 6 d before the tracer studies. Two subjects received [13C]AAA intravenously and 2 received it orally; 3 subjects received [13C]lysine intravenously and 6 received it orally. We measured 13CO2 output, plasma [13C]AAA and [13C]lysine enrichment, and urinary [13C]AAA. RESULTS: [13C]AAA oxidation was estimated to be higher after the orally administered than after the intravenously administer tracer; plasma [13C]AAA was similar to urinary [13C]AAA. Whole-body lysine oxidation showed a rhythm that was induced by meal feeding. The intravenous [13C]lysine tracer gave mean estimates of lysine balances (lysine intake minus oxidation) that apparently were too low (-15.7 mg x kg(-1) x d(-1)) or too high (16.6 mg x kg(-1) x d(-1), P < 0.05 from zero balance) on the basis of urinary [13C]AAA or plasma [13C]lysine estimates of oxidation, respectively. For the orally administered tracer and plasma [13C]lysine enrichment, the mean balance was slightly positive (8.7 mg x kg(-1) x d(-1), P < 0.05 from zero). CONCLUSIONS: Use of urinary [13C]AAA as an index of the enrichment of the precursor pool did not appear to significantly improve the estimate of the fasting and feeding components of daily lysine balance. For estimates of daily, whole-body lysine oxidation, we propose use of plasma [13C]lysine with a 24-h, orally administered tracer protocol.

A novel antifolate, MX-68, inhibits the development of autoimmune disease in MRL/lpr mice.

We compared a novel unpolyglutamable antifolate, MX-68, with polyglutamable antifolate, methotrexate (MTX), for treatment of an autoimmune kidney disease which develops spontaneously in MRL/Mp-lpr/lpr (MRL/lpr) mice. Oral administration of either MX-68 or MTX was commenced in 8-week-old female mice and continued 3 times a week until they reached 30 weeks of age. MX-68 delayed the onset of proteinuria and prolonged life span dose-dependently. Furthermore, it suppressed the elevation of serum blood urea nitrogen and cholesterol levels. MX-68 was as effective as MTX at ameliorating events which accompany the development of lupus nephritis, despite that MX-68 did not undergo polyglutamation. These ameliorative effects of MX-68 and MTX did not occur via inhibition of either autoantibody production or cell proliferation. Neither compound suppressed age-dependent elevation of immune complexes or antibodies for single-stranded DNA and TNP in serum nor did they influence the associated enlargement of lymph nodes and spleen. We conclude that MX-68 is beneficial for the treatment of autoimmune kidney disease in mice and may be useful for other related diseases such as systemic lupus erythematosus.

Selective ablation of astrocytes by intracerebral injections of alpha-aminoadipate.

The efficacy and the specificity of the putative astrotoxin, alpha-aminoadipate, were examined in this study. The integrity of astrocytes was evaluated at several time points following a single injection of alpha-aminoadipate into amygdala of adult rats using immunohistochemistry. The density and the morphological appearance of neurons and the response of microglia were also examined. The injection of alpha-aminoadipate disrupted the astrocytic network in that region. There was a profound loss of glial fibrillary acidic protein-positive and S100 beta-positive astrocytes, normally present in the region, while vimentin immunohistochemistry revealed the presence of deformed cell processes, presumably astrocytic. The presence of reactive microglia at the injection site was suggestive of an active degenerative process, while the normal neuronal density and appearance, as compared to controls, suggested that the damage was confined to astrocytes. The confirmed effectiveness and cellular specificity of alpha-aminoadipate in vivo makes it a potentially important experimental tool for attempting to decipher the functional significance of astrocytes.

Müller cell involvement in methanol-induced retinal toxicity.

Methanol is an ocular toxicant which causes visual dysfunction often leading to blindness after acute exposure. The physiological and biochemical changes responsible for this toxicity are poorly understood. Previously, we reported that the folate-reduced (FR) rat is an animal model which mimics the characteristic human methanol toxicities. The present study examines the hypothesis that depletion of ATP after methanol administration is the initiating event in methanol-induced retinal toxicity. ATP is reduced in retinae of methanol-treated FR rats to the same extent as is seen in retinae of FR and folate-sufficient (FS) rats treated with the Müller cell (retinal glial cell) toxin alpha-aminoadipic acid. Changes in the electroretinogram and the response of Müller cells to a potassium stimulus are also similarly eliminated in methanol-treated FR rats and alpha-aminoadipic acid-treated FR and FS rats. These results suggest that the Müller cell may be the initial target in methanol-induced visual system toxicity.

Contribution of glial cells to histamine inactivation.

DL-alpha-aminoadipic acid (DL-alpha AA), a selective gliotoxic agent produced significant reductions in histamine-N-methyl-transferase (HNMT) and monoamine oxidase-B (MAO-B) activities and an enhancement in histamine (HA) level in the hypothalamus of rats 2 and 4 h after single intracerebroventricular (i.c.v.) or subcutaneous (s.c.) injections of the compound. Histidine decarboxylase (HD) and monoamine oxidase-A (MAO-A) were unaffected after these treatments. Following a single i.c.v. injection of DL-alpha AA of 200 micrograms/rat, or a single s.c. injection of 5 mg/rat, marked diminutions in the astrocytic marker glutamine synthetase (GS) activity occurred suggesting marked glial damage in the hypothalamus. In total, these studies indicate an important role for glial cells in HA metabolism (inactivation).

Aminoadipic acid toxic effects on retinal glial cells.

The toxic effects of DL-alpha-aminoadipic acid on retinal glial cells were examined histologically following intravitreal injection to rats or rabbits. There was a marked swelling Müller cells, astrocytes and oligodendrocytes, but no changes in the microglia. Injections of a low concentration of the solution (0.1 mg in 0.01 ml) produced no irreversible damage to the Müller cells. With high concentrations (1 mg in 0.01 ml), there was a necrosis of Müller cells followed by thinning of the retina and disruption of the outer limiting membrane,

Scanning electron microscopy of retinal ganglion cells by applying D,L-2-aminoadipic acid.

Retinal ganglion cells of the cat were clearly demonstrated in scanning electron microscopy. For identification of the cells, D,L-2-aminoadipic acid, a gliotoxic compound, was applied. Müller cells as well as astrocytes were destroyed by intravitreal injection of the aminoadipic acid prior to the enucleation. Balloon-shaped retinal ganglion cells with an axonal process were visualized in the normal cat retina. Flattened and sometimes indented ganglion cells were observed in the pathologic retina which was made 2 weeks after optic nerve transection. Pretreatment with the aminoadipic acid was a very effective method for the identification of the retinal ganglion cells by scanning electron microscopy.