Jianhong An

Local Glutamate Level Dictates Adenosine A2A Receptor Regulation of Neuroinflammation and Traumatic Brain Injury

During brain injury, extracellular adenosine and glutamate levels increase rapidly and dramatically. We hypothesized that local glutamate levels in the brain dictates the adenosine–adenosine A2A receptor (A2AR) effects on neuroinflammation and brain damage outcome. Here, we showed that, in the presence of low concentrations of glutamate, the A2AR agonist 3-[4-[2-[[6-amino-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-oxolan-2-yl]purin-2-yl]amino]ethyl]phenyl]propanoic acid (CGS21680) inhibited lipopolysaccharide (LPS)-induced nitric oxide synthase (NOS) activity of cultured microglial cells, an effect that was dependent on the protein kinase A (PKA) pathway. However, in high concentrations of glutamate, CGS21680 increased LPS-induced NOS activity in a protein kinase C (PKC)-dependent manner. Thus, increasing the local level of glutamate redirects A2AR signaling from the PKA to the PKC pathway, resulting in a switch in A2AR effects from antiinflammatory to proinflammatory. In a cortical impact model of traumatic brain injury (TBI) in mice, brain water contents, behavioral deficits, and expression of tumor necrosis factor-α, interleukin-1 mRNAs, and inducible NOS were attenuated by administering CGS21680 at post-TBI time when brain glutamate levels were low, or by administering the A2AR antagonist ZM241385 [4-(2-{[5-amino-2-(2-furyl)[1,2,4]triazolo[1,5-a][1,3,5]triazin-7-yl]amino}ethyl)phenol] at post-TBI time when brain glutamate levels were elevated. Furthermore, pre-TBI treatment with the glutamate release inhibitor (S)-4C3HPG [(S)-4-carboxy-3-hydroxyphenylglycine] converted the debilitating effect of CGS21680 administered at post-TBI time with high glutamate level to a neuroprotective effect. This further indicates that the switch in the effect of A2AR activation in intact animals from antiinflammatory to proinflammatory is dependent on glutamate concentration. These findings identify a novel role for glutamate in modulation of neuroinflammation and brain injury via the adenosine–A2AR system.

Chronic but not acute treatment with caffeine attenuates traumatic brain injury in the mouse cortical impact model

Caffeine, the most consumed psychoactive drug and non-specific adenosine receptor antagonist, has recently been shown to exert a neuroprotective effect against brain injury in animal models of Parkinsons disease (PD) and stroke. However, the effects of caffeine on traumatic brain injury (TBI) are not known. In this study, we investigated the effects of acute and chronic caffeine treatment on brain injury in a cortical-impact model of TBI in mice. Following TBI, neurological deficits, cerebral edema, as well as inflammatory cell infiltration were all significantly attenuated in mice pretreated chronically (for 3 weeks) with caffeine in drinking water compared with the mice pretreated with saline. Furthermore, we found that chronic caffeine treatment attenuated glutamate release and inflammatory cytokine production, effects that were correlated with an upregulation of brain A1 receptor mRNA. By contrast, acute treatment with caffeine (i.p. injection, 30 min before TBI) was not effective in protecting against TBI-induced brain injury. These results suggest that chronic (but not acute) caffeine treatment attenuates brain injury, possibly by A1 receptor-mediated suppression of glutamate release and inhibition of excessive inflammatory cytokine production. These results highlight the potential benefit of chronic caffeine intake for preventing TBI and provide a rationale for the epidemiological investigation of the potential association between TBI and human caffeine intake.

Genetic inactivation of adenosine A2A receptors attenuates acute traumatic brain injury in the mouse cortical impact model

The inactivation of the A(2A) receptor (A(2A)R) has been shown to neuroprotect against brain injury in several animal models of neurological disorders including stroke and Parkinsons disease. However, despite marked elevation of adenosine level, the role of the A(2A) in traumatic brain injury (TBI) remains unclear. In the present study, we investigated the effects of genetic inactivation of A(2A)Rs in the acute stage. The A(2A)R knock-out (KO) mice and their wild-type (WT) littermates were subjected to cortical impact injury by a dropping weight. The control group was only craniotomized without TBI. At 24 h post-TBI, the neurological deficit scores of the KO mice were significantly lower than that of WT littermates. Consistent with the behavioral changes, the brain water contents as well as histological changes and the TUNEL-positive cells of the injured cortex of the KO mice were significantly lower than that of WT littermates. Furthermore, the glutamate level in the cerebral spinal fluid (CSF) of the KO mice was also significantly lower than that of WT littermates. In addition, we found that at 12 h post-TBI the mRNA and protein levels of TNF-alpha and IL-1beta were higher in the KO mice than that in the WT littermates. However, at 24 h post-TBI, the level of TNF-alpha and IL-1beta continually increased in the WT mice but largely declined in the KO mice. These results suggest that the genetic inactivation of A(2A)R protects against TBI, which is mainly associated with the suppression of glutamate level.

Plasma glutamate–modulated interaction of A2AR and mGluR5 on BMDCs aggravates traumatic brain injury–induced acute lung injury

Activation of adenosine A2A receptor aggravates lung damage in a neurogenic mouse model of acute lung injury (ALI) but protects against nonneurogenic ALI.

Genetic deletion of the adenosine A2A receptor confers postnatal development of relative myopia in mice.

PURPOSE To critically evaluate whether the adenosine A2A receptor (A2AR) plays a role in postnatal refractive development in mice. METHODS Custom-built biometric systems specifically designed for mice were used to assess the development of relative myopia by examining refraction and biometrics in A2AR knockout (KO) mice and wild-type (WT) littermates between postnatal days (P)28 and P56. Ocular dimensions were measured by customized optical coherence tomography (OCT), refractive state by eccentric infrared photorefraction (EIR), and corneal radius of curvature by modified keratometry. Scleral collagen diameter and density were examined by electron microscopy on P35. The effect of A2AR activation on collagen mRNA expression and on soluble collagen production was examined in cultured human scleral fibroblasts by real-time RT-PCR and a collagen assay kit. RESULTS Compared with WT littermates, the A2AR KO mice displayed relative myopia (average difference, 5.1 D between P28 and P35) and associated increases in VC depth and axial length from P28 to P56. Furthermore, the myopic shift in A2AR KO mice was associated with ultrastructural changes in the sclera: Electron microscopy revealed denser collagen fibrils with reduced diameter in A2AR KO compared with WT. Last, A2AR activation induced expression of mRNAs for collagens I, III, and V and increased production of soluble collagen in cultured human scleral fibroblasts. CONCLUSIONS Genetic deletion of the A2AR promotes development of relative myopia with increased axial length and altered scleral collagen fiber structure during postnatal development in mice. Thus, the A2AR may be important in normal refractive development.

Adenosine A2A receptors in both bone marrow cells and non-bone marrow cells contribute to traumatic brain injury

J. Neurochem. (2010) 113, 1536–1544.

Activation of dopamine D2 receptor is critical for the development of form-deprivation myopia in the C57BL/6 mouse.

PURPOSE This study used dopamine D2 receptor (D2R) knockout (KO) mice to investigate the role of D2R activity in the development of form-deprivation myopia (FDM). Sulpiride, a D2R antagonist, was administered systemically into wild-type (WT) mice to validate the involvement of D2R in FDM development. METHODS The D2R KO and WT C57BL/6 mice were subjected to FDM. Wild-type mice received daily intraperitoneal injections of sulpiride, 8 μg/g body weight, for a period of 4 weeks. The body weight, refraction, corneal radius of curvature, and ocular axial components were measured at week 4 of the experiment. Differences in all ocular parameters between the experimental and control groups were compared statistically. RESULTS Form-deprivation myopia in D2R KO mice (FD-KO) was significantly reduced compared with their WT littermates (interocular difference, -2.12 ± 0.91 diopter [D] in FD-KO versus -5.35 ± 0.83 D in FD-WT, P = 0.014), with a smaller vitreous chamber depth (0.008 ± 0.006 vs. 0.026 ± 0.006 mm, P = 0.044) and axial length (-0.001 ± 0.007 vs. 0.027 ± 0.008 mm, P = 0.007). Furthermore, FDM was attenuated in animals treated with sulpiride (-2.01 ± 0.31 D in FD-sulpiride versus -4.06 ± 0.30 D in FD-DMSO, P < 0.001) compared with those treated with vehicle, with a retardation in growth of vitreous chamber depth (-0.001 ± 0.006 vs. 0.022 ± 0.004 mm, P = 0.003) and axial length (-0.004 ± 0.007 vs. 0.027 ± 0.005 mm, P = 0.001). CONCLUSIONS Genetic and pharmacological inactivation of D2R attenuates FDM development in mice, suggesting that dopamine acting on D2R appears to promote the development of FDM in C57BL/6 mice. Further studies are required to confirm these results using animal models in which retinal D2R is selectively blocked.

A Comparative Transcriptomic Analysis of Uveal Melanoma and Normal Uveal Melanocyte

Background Uveal melanoma is the most common primary intraocular tumor in adults in western countries. It is associated with very severe visual morbidity and may lead to distant metastases even after successful treatment of the primary tumor. In order to gain better insight into molecular mechanisms related to tumorigenesis and metastasis of uveal melanoma, we used next-generation sequencing technology (SOLiD, Life Technologies) to acquire global transcriptome alteration between posterior uveal melanoma cells and normal uveal melanocyte. Results From mRNAs of the cultured uveal melanoma cells and normal uveal melanocytes, we annotated more than 3.7×107 and 2.7×107 sequencing tags based on human Ensembl databases, respectively. For detailed analysis, we chose 5155 well-annotated genes mainly involved in the MAPK signaling pathway, cell cycle, cell adhesion junction, apoptosis, and P53 signaling pathways as well as melanogenesis. In an effort to confirm the authenticity of our sequencing results, we validated twenty-one identically differentially expressed genes by using quantitative real time PCR from cultured cell lines of other posterior uveal melanoma cells and normal uveal melanocytes. Conclusion We have identified a large number of potentially interesting genes for biological investigation of uveal melanoma. The expression profiling also provides useful resources for other functional genomic and transcriptome studies. These 21 potential genes could discriminate between uveal melanoma cells and normal uveal melanocyte, which may be indicative of tumorigenesis process. Our results further suggest that high-throughput sequencing technology provides a powerful tool to study mechanisms of tumogenesis in the molecular level.

cAMP Level Modulates Scleral Collagen Remodeling, a Critical Step in the Development of Myopia

The development of myopia is associated with decreased ocular scleral collagen synthesis in humans and animal models. Collagen synthesis is, in part, under the influence of cyclic adenosine monophosphate (cAMP). We investigated the associations between cAMP, myopia development in guinea pigs, and collagen synthesis by human scleral fibroblasts (HSFs). Form-deprived myopia (FDM) was induced by unilateral masking of guinea pig eyes. Scleral cAMP levels increased selectively in the FDM eyes and returned to normal levels after unmasking and recovery. Unilateral subconjunctival treatment with the adenylyl cyclase (AC) activator forskolin resulted in a myopic shift accompanied by reduced collagen mRNA levels, but it did not affect retinal electroretinograms. The AC inhibitor SQ22536 attenuated the progression of FDM. Moreover, forskolin inhibited collagen mRNA levels and collagen secretion by HSFs. The inhibition was reversed by SQ22536. These results demonstrate a critical role of cAMP in control of myopia development. Selective regulation of cAMP to control scleral collagen synthesis may be a novel therapeutic strategy for preventing and treating myopia.

Adenomatous Polyposis Coli Mutation Leads to Myopia Development in Mice

Myopia incidence in China is rapidly becoming a very serious sight compromising problem in a large segment of the general population. Therefore, delineating the underlying mechanisms leading to myopia will markedly lessen the likelihood of other sight compromising complications. In this regard, there is some evidence that patients afflicted with familial adenomatous polyposis (FAP), havean adenomatous polyposis coli (APC) mutation and a higher incidence of myopia. To clarify this possible association, we determined whether the changes in pertinent biometric and biochemical parameters underlying postnatal refractive error development in APCMin mice are relevant for gaining insight into the pathogenesis of this disease in humans. The refraction and biometrics in APCMin mice and age-matched wild-type (WT) littermates between postnatal days P28 and P84 were examined with eccentric infrared photorefraction (EIR) and customized optical coherence tomography (OCT). Compared with WT littermates, the APCMin mutated mice developed myopia (average -4.64 D) on P84 which was associated with increased vitreous chamber depth (VCD). Furthermore, retinal and scleral changes appear in these mice along with: 1) axial length shortening; 2) increased retinal cell proliferation; 3) and decreased tyrosine hydroxylase (TH) expression, the rate-limiting enzyme of DA synthesis. Scleral collagen fibril diameters became heterogeneous and irregularly organized in the APCMin mice. Western blot analysis showed that scleral alpha-1 type I collagen (col1α1) expression also decreased whereas MMP2 and MMP9 mRNA expression was invariant. These results indicate that defective APC gene function promotes refractive error development. By characterizing in APCMin mice ocular developmental changes, this approach provides novel insight into underlying pathophysiological mechanisms contributing to human myopia development.