Mapping P2X and P2Y receptor proteins in striatum and substantia nigra: An immunohistological study

Our work aimed to provide a topographical analysis of all known ionotropic P2X1–7 and metabotropic P2Y1,2,4,6,11–14 receptors that are present in vivo at the protein level in the basal ganglia nuclei and particularly in rat brain slices from striatum and substantia nigra. By immunohistochemistry-confocal and Western blotting techniques, we show that, with the exception of P2Y11,13 receptors, all other subtypes are specifically expressed in these areas in different amounts, with ratings of low (P2X5,6 and P2Y1,6,14 in striatum), medium (P2X3 in striatum and substantia nigra, P2X6,7 and P2Y1 in substantia nigra) and high. Moreover, we describe that P2 receptors are localized on neurons (colocalizing with neurofilament light, medium and heavy chains) with features that are either dopaminergic (colocalizing with tyrosine hydroxylase) or GABAergic (colocalizing with parvalbumin and calbindin), and they are also present on astrocytes (P2Y2,4, colocalizing with glial fibrillary acidic protein). In addition, we aimed to investigate the expression of P2 receptors after dopamine denervation, obtained by using unilateral injection of 6-hydroxydopamine as an animal model of Parkinson’s disease. This generates a rearrangement of P2 proteins: most P2X and P2Y receptors are decreased on GABAergic and dopaminergic neurons, in the lesioned striatum and substantia nigra, respectively, as a consequence of dopaminergic denervation and/or neuronal degeneration. Conversely, P2X1,3,4,6 on GABAergic neurons and P2Y4 on astrocytes augment their expression exclusively in the lesioned substantia nigra reticulata, probably as a compensatory reaction to dopamine shortage. These results disclose the presence of P2 receptors in the normal and lesioned nigro-striatal circuit, and suggest their potential participation in the mechanisms of Parkinson’s disease.
Introduction
It is now well established that the arrangement of ionotropic P2X and metabotropic P2Y receptors [1, 2] on a cell membrane is a very dynamic process, often related to developmental or physiopathological conditions. Moreover, it is common knowledge that multiple P2 proteins are simultaneously recruited on a cell membrane for triggering biological functions. As a consequence, P2 receptors are rightly considered more than the sum of their single entities and must be therefore regarded as a complex network of cooperating receptors. Under this perspective, a numerical model was also introduced, the combinatorial receptor web model, which explains the biological efficacy of combining an assorted array of different P2 proteins on a given cell, in order to integrate, upgrade, guarantee and optimize specific receptor-dependent functions [3].
This trend of course applies to the central nervous system (CNS) as well, where in situ hybridization of P2 mRNA subtypes and immunohistochemistry of P2 proteins shows, for instance, wide but heterogeneous simultaneous distribution of both P2X [4–11] and P2Y [12–15] classes of receptors. In particular, P2X2,4,6 and P2Y1 subtypes are abundant and widespread approximately in the entire brain, while P2X1 protein is enriched in the cerebellum, P2X3 in the brain stem, and P2X7 is largely prejunctional. The hippocampus concurrently expresses all P2X and, moreover, P2Y1,2,4,6,12 receptor subtypes. Particularly in the basal ganglia (BG), neostriatal medium-spiny neurons and cholinergic interneurons highly express P2X2 and P2Y1 receptors, but it appears that they become functional only under certain, as yet unknown, conditions [16]. Moreover, P2X2 receptor protein was described in substantia nigra pars compacta (SNC) [17], whereas both protein and mRNA were described in SNC and striatum [18]. Finally, only very low levels of P2X4,6 mRNAs were detected in substantia nigra (SN) and striatum [19].
By functional analysis, ATP release was demonstrated from cultured embryonic neostriatal neurons [20], and ATP-evoked potassium currents in rat striatal neurons were shown to be mediated by P2 receptors [21]. ATP was also proved to increase extracellular dopamine levels in rat striatum through stimulation of P2Y subtypes [22], although it was claimed to inhibit dopamine release in the neostriatum [23]. Extracellular ATP via P2 receptors was finally reported to induce neurotoxicity in vitro [24] and in vivo [25] in the striatum. Besides P2 receptors on neurons, in BG there is also evidence of P2 receptors on, and release of ATP from, glial cells. P2Y12 subtype is present, for instance, on oligodendrocytes in striatum and SN [26], and P2X7 receptor is upregulated on microglia in striatum after middle cerebral artery occlusion [27]. In spite of these results, there is a general paucity of studies addressing the cellular distribution of all P2 receptor proteins in BG.
Our work thus aimed to provide the complete topographical analysis of known P2X and P2Y subtypes that are present in rat striatum and SN in vivo, and to investigate the dynamic presence of P2 proteins after the induction of experimental parkinsonism by dopamine-denervation achieved by using the unilateral 6-hydroxydopamine (6-OHDA) rat model. By upgrading the current map of P2 receptors expressed in the brain, our study discloses the potential impact of these receptors in the normal and lesioned nigro-striatal circuit.
Materials and methods
Histological procedures
Wistar rats (Harlan, Udine, Italy) were anesthetized by i.p. injections of sodium pentobarbital (60 mg/kg), and transcardially perfused with saline (0.9 % NaCl) followed by 4% paraformaldehyde, in phosphate buffer (PB, 0.1 M pH 7.4). Each brain was immediately removed, post-fixed in the same fixative for 2 h, and then transferred to 30% sucrose in PB at 4°C, until it sank. The experimental protocol used in this study was approved by the Italian Ministry of Health and was in agreement with the guidelines of the European Communities Council Directive of November 24, 1986 (86/609/EEC) for the care and use of laboratory animals. All efforts were made to minimize the number of animals used and their suffering.
Double immunofluorescence
Transverse sections (40-μm thick) were cut on a freezing microtome and were processed for double immunofluorescence studies. Non-specific binding sites were blocked with 10% normal donkey serum in 0.3% Triton X-100, in phosphate buffered saline (PBS) for 30 min at room temperature. The sections were incubated in a mixture of primary antisera for 24–48 h in 0.3% Triton X-100 in PBS. Rabbit anti-P2r (1:300, Alomone, Jerusalem, Israel) was used in combination with either mouse anti-calbindin-D-28K (1:200, Sigma, Mi, Italy), mouse anti-tyrosine hydroxylase (TH, 1:500, Sigma), mouse anti-parvalbumin (1:200, Chemicon International, Temecula, CA, USA), mouse anti-glial fibrillary acidic protein (GFAP) (1:400, Sigma), mouse anti-myelin basic protein (MBP, 1:200, Chemicon International), mouse anti-neurofilament H non-phosphorylated (SMI 32, 1:500, Sternberger Monoclonals, Lutherville, MD, USA), mouse anti-neurofilament H and M non-phosphorylated (SMI 33, 1:500, Sternberger Monoclonals), mouse anti-neurofilament 160 (NF160, 1:500, Sigma) or goat anti-neurofilament-L protein (NF-L, 1:100, Santa Cruz, Mi, Italy). The secondary antibodies used for double labeling were Cy3-conjugated donkey anti-rabbit IgG (1:100, red immunofluorescence, Jackson Immunoresearch, West Baltimore Pike, PA, USA), Cy2-conjugated donkey anti-mouse IgG (1:100, green immunofluorescence, Jackson Immunoresearch) or Cy2-conjugated donkey anti-goat IgG (1:100, green immunofluorescence, Jackson Immunoresearch).
The sections were washed in PBS three times for 5 min each, and then incubated for 3 h in a solution containing a mixture of the secondary antibodies in 1% normal donkey serum in PBS. After rinsing, the sections were mounted on slide glasses, allowed to air dry and coverslipped with gel/mount anti-fading medium (Biomeda, Foster City, CA, USA).
Confocal microscopy
Double- or triple-label immunofluorescence was analyzed by means of a confocal laser scanning microscope (CLSM) (LSM 510, Zeiss, Arese, Mi, Italy) equipped with argon laser emitting at 488 nm, helium/neon laser emitting at 543 nm, and helium/neon laser emitting at 633 nm. Specificity of the antibodies was positively proved by performing confocal analysis in the absence of the primary antibodies, but in the presence of either anti-rabbit or anti-mouse secondary antibodies. Specificity was further confirmed for the P2r antiserum by performing immunoreactions in the simultaneous presence of the P2r neutralizing immunogenic peptides.
Isolation of cerebral areas and protein extraction
Wistar rats were anesthetized by i.p. injections of sodium pentobarbital (60 mg/kg) and, after decapitation, brains were removed. Each brain was transversally cut on a vibratome (300 μm). The specific cerebral areas were isolated with the aid of a dissection microscope and homogenized in RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS in PBS containing protease inhibitors). After short sonication, the homogenates were incubated on ice for 1 h and centrifuged at 14,000 r.p.m. for 10 min at 4°C. Protein quantification was performed in the supernatants by Bradford colorimetric assay (Biorad, Milan, Italy).
Western blot analysis
Equal amounts of cell lysate (20–30 μg of protein from each cerebral area) were separated by electrophoresis on 10–12% SDS-PAGE and transferred to nitrocellulose membranes Hybond-C extra (Amersham Biosciences, Cologno Monzese, Italy). The filters were pre-wetted in 5% non-fat milk in TBS-T (10 mM Tris pH 8, 150 mM NaCl, 0.1% Tween 20) and hybridized overnight with P2X1,2,4 antisera (Alomone, 1:500), P2X5 and P2Y4/14 (1:200), P2Y6 (1:300) or P2Y2 (1:400). The antisera were immunodetected with an anti-rabbit HRP-conjugated antibody (1:5,000) and developed by ECL chemiluminescence (Amersham Biosciences), using Kodak Image Station (KDS IS440CF).
Anti-P2r specificity
The polyclonal P2r antisera used in this study were raised against P2r highly purified peptides (identity confirmed by mass spectrography and amino acid analysis, as indicated in the certificate of analysis provided by the manufacturer), corresponding to specific epitopes not present in any other known protein. The specificity of the P2r signals was moreover assessed by incubating Western blots either in the absence of the primary antiserum, or in the presence of the primary antiserum together with the neutralizing P2r immunogenic peptides (μg protein ratio 1:1 between peptide and antiserum).
6-OHDA lesion and Nissl staining
Deeply anesthetized rats (45 days old, about 150 g body weight) were injected with 8 μg/4 μl 6-OHDA in saline 0.1% ascorbic acid in the medial forebrain bundle (stereotaxic coordinates ap = −4.4; l = +1.2; vd = −7.8, see also Paxinos et al. [28]) at a rate of 0.38 μl/min. Fifteen days later, the lesioned rats were tested with 0.05 mg/kg s.c. of the D1/D2 dopamine agonist apomorphine, in order to verify the efficacy of the 6-OHDA lesion, and contralateral turns to the lesion were counted for 40 min. Only those rats that made at least 200 contralateral turns were used for the study. It has been previously demonstrated that rats meeting this screening criterion have greater than 95% depletion of striatal dopamine [29]. At 1.5 months after the 6-OHDA lesion, rats were used for immunohistological experiments (n = 3). In order to evaluate cell damage, 40-μm rat brain sections were mounted onto gelatinized slides. They were dehydrated through alcohols, and then rehydrated and stained in 2% cresyl violet for 45 min. Following deionized water rinses, the slides were dehydrated in a standard alcohol series, cleared in xylene, and coverslipped.
Results
P2X and P2Y receptor proteins in rat striatum
We describe in this work the cellular and subcellular in vivo distribution of P2X and P2Y receptors in transverse sections of adult rat striatum, showing by double immunofluorescence confocal analysis that the various P2 subtype proteins are distinguished by different degrees of expression and are not uniformly distributed throughout the entire tissue (Fig. 1).
Fig. 1P2X and P2Y receptor proteins in rat striatum. Transverse sections through the striatum of adult rats were processed for double immunofluorescence studies. Rabbit polyclonal antisera against P2X1,2,4 and P2Y4 receptors (red Cy3 immunofluorescence) were used in combination with antibodies against neuronal or glial markers (green Cy2 immunofluorescence). Panel A P2X1: confocal images illustrate clear colocalization of P2X1 receptor with neurofilament-light protein (NF-L). The merged field of inset a shows absence of colocalization between the neuronal GABAergic marker calbindin (green) (a calcium-binding protein expressed mainly in medium spiny neurons of the striatum) and P2X1 receptor (red). The merged field of inset b shows the merged field of P2X1 (red) and MBP (green) overlapping immunoreactivities at higher magnification. Panel B P2X2: double immunofluorescence demonstrates that P2X2 receptor immunoreactivity (red) colocalizes with calbindin protein (green). The insets e and f show colocalization with the neuronal GABAergic marker parvalbumin (green) (a calcium-binding protein that is expressed in interneurons of the striatum). Panel C P2X4: red immunofluorescence for P2X4 protein merges with the green signals of the three types of neurofilament proteins: NF-L (inset g, merged field), NF160 (inset i, merged field) and SMI 32 and, moreover, with parvalbumin (inset h, merged field) and calbindin (inset i, merged field). Panel D P2Y4: red P2Y4 immunoreactivity is present on calbindin-positive neurons (green), on parvalbumin-positive neurons (green) (insets n–q), and on GFAP-positive astrocytes (inset o, merged field). Western blot analysis also confirms the presence of receptor proteins P2X1,2,4 (insets c in panel A, d in panel B, m in panel C, respectively) and P2Y4 (inset r in panel D) in striatum. Specificity of the P2 receptor signals was assessed by incubations of the primary antisera with the corresponding neutralizing immunogenic peptides (μg protein ratio 1:1 between peptide and antiserum). Scale bars are 10 μm in A; 100 μm in inset a; 2 μm in inset b; 50 μm in B and in insets e and f; 20 μm in C; 10 μm in insets h, i, l; 5 μm in inset g; 50 μm in D; 5 μm in inset n; and 20 μm in inset o. Similar results were obtained in at least four independent experiments
In particular, a strong P2X1 receptor immunoreactivity (red) confers a patchy appearance to the striatum, being localized mainly in white matter, while sparing the projecting calbindin-positive GABAergic neurons that are highly enriched in gray matter (Fig. 1A, inset a, green). Moreover, P2X1 protein immunofluorescence is present on NF-L positive, transversally oriented neuronal fibers, although the merged field provides only partial colocalization between the two signals (Fig. 1A). In addition, the high magnification analysis (Fig. 1A, inset b) of P2X1 (red) and MBP (green) immunoreactive signals shows that P2X1 receptor is surrounded by MBP, proving the presence of P2X1 protein on myelinated fibers. Due to the close vicinity of the two signals, overlapping yellow immunofluorescence is also observed. Finally, P2X1 receptor in striatum is recognized by Western blot analysis as a single protein band of 60–65 kDa, additionally abolished in the presence of the P2X1 receptor–neutralizing immunogenic peptide (Fig. 1A, inset c).
Conversely, an abundant P2X2 receptor immunoreactivity (red) is found in gray matter of striatum (Fig. 1B), while sparing the bundles of white matter. Specific receptor immunolabeling is present not only on the highly expressed calbindin-positive projecting GABAergic neurons, but also on the fewer parvalbumin-positive GABAergic interneurons (Fig. 1B, insets e, f). By Western blot analysis, we show that P2X2 receptor is present in striatum under two isoforms of about 45 and 32 kDa, furthermore, it is abolished in the presence of the P2X2 receptor–neutralizing immunogenic peptide (Fig. 1B, inset d).
P2X3 receptor immunostaining in striatum is of medium intensity (Table 1), and mainly localizes on GABAergic neurons of gray matter (data not shown).
Table 1Map of P2 receptor proteins in striatum and substantia nigra StriatumSubstantia nigraP2X1++++++P2X2++++++P2X3++++P2X4++++++P2X5++++P2X6+++P2X7++++P2X1+++P2X2++++++P2X4++++++P2X6++++P2X11––P2X12++++++P2X13––P2X14–+++Relative abundance of all P2X and P2Y receptor proteins was analyzed by confocal immunofluorescence microscopy, as described in “Materials and methods”. The intensity of the specific immunostaining was scored as follow: – = not detected; + = just sufficient to evaluate presence and outline of positive cells; ++ = adequate to assess morphological features of cell bodies and/or cellular processes; +++ = very bright
P2X4 receptor signal is instead very copious in white matter, although present on a few fibers of gray matter as well (Fig. 1C, red). It partially colocalizes with all types of heavy-, light- and medium-chain neurofilament proteins (merged fields): SMI 32 (green), NF-L (inset g), and NF160 (inset i). Moreover, we find P2X4 protein also on GABAergic interneurons (inset h) and GABAergic spiny neurons (inset l). By Western blot analysis, we demonstrate that P2X4 receptor is present in striatum as a single band of about 60 kDa, moreover, it is abolished in the presence of the P2X4 receptor–neutralizing immunogenic peptide (Fig. 1C, inset m).
P2X5,6,7 and P2Y1 receptor immunoreactivities in striatum are very weak (Table 1) in gray matter, although totally absent from white matter under our experimental conditions (data not shown).
The P2Y2 receptor is highly expressed in striatum on axons of white matter and astrocytes of gray matter (Table 1). Moreover, it is detected as a double protein band in the 55–65 kDa range (data not shown).
A strong P2Y4 receptor immunoreactivity is present only in gray matter of striatum, localized on both types of GABAergic neurons: calbindin-positive (Fig. 1D) and parvalbumin-positive (insets n–q). Nevertheless, the receptors are also widespread throughout the striatum on astrocytes, as shown by colocalization with the GFAP marker (inset o). By Western blot analysis, we prove that P2Y4 receptor is present in striatum as a double band of about 42 and 85 kDa (inset r), likely corresponding to the monomeric and dimeric aggregation states of the receptor [30, 31].
While P2Y6 receptor is barely detectable (Table 1) on GABAergic neurons in striatum (data not shown), P2Y11,13,14 receptor proteins were not identified by any means under our experimental conditions (Table 1). Finally, P2Y12 receptor in striatum (Table 1) is abundantly expressed only on oligodendrocytes and myelin sheets, as previously shown [26].
P2X and P2Y receptor proteins in substantia nigra
We conducted a parallel analysis on the cellular and subcellular in vivo distribution of P2X and P2Y receptors in transverse sections of adult rat SN. We showed by double immunofluorescence confocal analysis that the different P2 receptor proteins possess more comparable levels of expression with respect to the striatum, and are also more uniformly, although differently, distributed throughout the entire SNC and SNR (Fig. 2). In particular, strong signals for ionotropic P2X2,5 (red, Fig. 2A,B), P2X1,4 (Table 1), metabotropic P2Y6,14 (red, Fig. 2C,D) and P2Y4 (Table 1), or moderate signals for P2X3,6 and P2Y1 receptors (Table 1) are present on dopaminergic neurons (TH-positive) of SNC. Moreover, P2Y2 and P2Y12 receptors are abundantly expressed in SN (Table 1), but P2Y2 is expressed on axons and astrocytes, and P2Y12 only on oligodendrocytes and myelin sheets [26]. Conversely, in SNR, a weak P2X/Y receptor immunoreactivity is limited to sparse neuronal bodies, likely identified as GABAergic neurons by colocalization with parvalbumin (data not shown). The presence at the tissue level in SN of ionotropic P2X2,5 (insets a in panel A, and b in panel B of Fig. 2, respectively) and metabotropic P2Y6,14 (insets c in panel C, and d in panel D of Fig. 2, respectively) proteins is confirmed by Western blot analysis performed in all cases in the presence of specific receptor–neutralizing immunogenic peptides. Similarly to the striatum, immunoreactive signals for P2Y11,13 receptors were not identified by any means under our experimental conditions (Table 1).
Fig. 2P2X and P2Y receptor proteins in rat substantia nigra. Double immunofluorescence visualized by confocal analysis was performed in transverse sections through the substantia nigra of adult rats. Strong signals for ionotropic P2X2,5 and metabotropic P2Y6,14 (red Cy3 immunofluorescence) are present on dopaminergic neurons (TH-positive, green Cy2 immunofluorescence) of substantia nigra pars compacta (SNC), whereas in substantia nigra pars reticolata (SNR) P2X/Y immunoreactivity is limited to sparse neuronal bodies. Western blot analysis confirms the presence in substantia nigra of receptor proteins P2X2,5 (insets a in panel A, and b in panel B, respectively) and P2Y6,14 (insets c in panel C, and d in panel D, respectively). Specificity of the P2 receptor signals was assessed by incubation of the primary antisera with the corresponding neutralizing immunogenic peptides (μg protein ratio 1:1 between peptide and antiserum).Scale bars in all panels are 50 μm. Similar results were obtained in at least four independent experiments
6-Hydroxydopamine modulates the expression of selected P2 receptors in striatum and substantia nigra
No contralateral rotation as a sign of motor deficit was reported in rats before being 6-OHDA-lesioned, but was instead detected after the lesion rotation (data not shown), together with loss of dopaminergic TH-positive neurons only from the ipsilateral hemisphere of SNC (Fig. 3A and insets a, b).
Fig. 36-Hydroxydopamine modulates the expression of selected P2 receptor proteins in striatum and substantia nigra. Staining of rat substantia nigra after 6-hydroxydopamine treatment. Panel A Conventional microscopy images of Nissl staining shows several dopaminergic neurons (arrows) in the contralateral control hemisphere, which are lost (asterisks) in the ipsilateral lesioned hemisphere. Specific ipsilateral dopaminergic lesion of substantia nigra pars compacta (SNC) was also visualized by confocal TH-immunostaining (green) (insets a, b). Panel B Confocal merged yellow images show upregulation of P2X1 receptor protein (red) in parvalbumin-positive GABAergic neurons (green) in the lesioned side of substantia nigra pars reticolata (SNR) of 6-hydroxydopamine-treated rats. Panel C Confocal merged yellow images show a drastic increase in GFAP-positive astrocytes (green) in the lesioned side of 6-hydroxydopamine-treated rats and, correspondingly, an augment of P2Y4 signal (red) (inset c). Scale bars are 100 μm in A, B and in insets a, b; 20 μm in C; and 10 μm in inset c. Similar results were obtained in at least three independent experiments
Concomitantly, we prove that dopamine denervation in the 6-OHDA-lesioned rat generates a significant and selective rearrangement of P2 receptor proteins. Whereas the expression pattern and immunofluorescence intensities of P2X1,4, P2Y2 (colocalizing with all neurofilaments and present in white matter on fibers projecting from the cortex), and P2Y12 (present on oligodendrocytes of white matter) remain constant in both ipsi- and contralateral hemispheres after 6-OHDA treatment (as well as in control animals), all other P2X and P2Y receptors are decreased on parvalbumin- and calbindin-positive GABAergic neurons of deafferented ipsilateral striatum (but not contralateral and in control animals), as measured by semiquantitative analysis (Table 2) (n = 3).
Table 2Map of P2 receptor modulation after dopamine denervation Ipsilateral Striatum Ipsilateral SNP2X1=↑GABAP2X2↓GABA↓THP2X3↓GABA↓TH, ↑GABAP2X4↓GABA↓TH, ↑GABAP2X5=↓THP2X6=↓TH, ↑GABAP2X7==P2X1=↓THP2X2==P2X4↓GABA↑GFAPP2X6=↓THP2X11==P2X12==P2X13==P2X14=↓THRelative increase (↑) or decrease (↓) in P2X and P2Y receptor proteins analyzed by confocal immunofluorascence microscopy in striatum and SN after treatment in rat in vivo with 6-hydroxydopamine (ipsilateral), and in control (not lesioned) brain hemisphere (contralateral).TH= presence in dopaminergic neurons,GABA= presence in GABAergic neurons,GFAP= presence in astrocytes
Similarly, all P2X and P2Y receptors are lost in the lesioned (but not contralateral) substantia nigra pars compacta, consequent to the degeneration of the majority of TH-positive dopaminergic neurons (Table 2). Conversely, P2X1 (Fig. 3B) and P2X3,4,6 (Table 2) receptors present on GABAergic neurons, and P2Y4 receptors on astrocytes augment their expression only in ipsilateral substantia nigra pars reticulata adjacent to the lesioned pars compacta. In this same area, a phenomenon of astrogliosis is also induced, as detected by more abundant expression of GFAP-positive astrocytes (Fig. 3C).
Discussion
Because the roles of ATP in the CNS have received less attention until recently, often due to lack of appropriate research tools, our knowledge of the functional qualification of P2 receptors in the brain is limited, although rapidly improving. As a group of nuclei interconnected with cerebral cortex, thalamus and brainstem, and associated with a variety of functions, such as motor control, cognition, emotions and learning, the BG [32] is an area that deserves thorough analysis. Our work was aimed at mapping in vivo the presence of P2 receptor subtypes in the BG nuclei of striatum and SN by immunofluorescence-confocal and Western blotting techniques. The specificity of the highly sensitive molecular probes used for the detection of all known P2X and P2Y receptor proteins has been previously validated [33, 34]. In addition, we undertook an analysis that excluded possible cross-reactivity for all antisera used.
Our results not only establish that the majority of P2X (P2X1–7) and P2Y (P2Y1,2,4,6,11–14) receptors so far cloned from mammalian tissues are found in striatum and SN, but also prove their distinctive localization on neurons and/or glial cells. In detail we show that, with the exception of only P2Y11 and P2Y13 receptors (whose immunoreactivity was not identified by any means under our experimental conditions), all other subtypes are specifically localized in striatum and SN (both pars compacta and reticulata), although with different levels of expression, rated as low (P2X5,6 and P2Y1,6,14 in striatum), medium (P2X3 in striatum and SN; P2X6,7 and P2Y1 in SN) and high. Moreover, while we show a prevalence of P2 receptors on neurons (P2X1,4 and P2Y2 colocalizing with neurofilament light, medium and heavy chains) with features that are either dopaminergic (P2X2–5 and P2Y1,4,6,14 colocalizing with TH, in SN) or GABAergic (P2X2–4 and P2Y4 colocalizing with parvalbumin and calbindin, in striatum), we also describe their expression on astrocytes (P2Y2,4 in striatum and SN, colocalizing with GFAP), microglia (P2X7, colocalizing with OX42) [27], and oligodendrocytes (P2Y12, colocalizing with MBP and RIP) [26]. By confirming previous autoradiographic studies [35, 36], our results therefore prove the widespread but diversified P2-receptor protein distribution in striatum and SN, and extend to these nuclei the great level of biological complexity and molecular sophistication pertaining to P2 receptors [3].
Although the configuration of receptor subunits required for assembly into functional cation channels gated by extracellular ATP in different regions of the CNS comprising the BG is not known yet, colocalization of so many different P2X subtypes in striatum and SN is definitely compatible with heteromultimeric assembly of ionotropic subunits. Since a growing body of biochemical and biophysical evidence now indicates that the propensity to form homo- and especially hetero-multimers is frequent also for G protein-coupled receptors [37] comprising the P2Y subtypes [30, 31], the concurrent expression in striatum and SN of as many metabotropic receptors could explain once more a complex hetero-oligomeric architecture. Nevertheless, the biological phenomenon of redundancy could also justify the simultaneous presence of multiple P2 receptor subtypes in these nuclei, with the final outcome of increasing the structural and pharmacological heterogeneity of these brain regions. Finally, the composite architecture of P2 receptors that we depicted in striatum and SN might likely also signify a multipart mechanism of receptor cooperative behavior (Volonté et al., personal communication) that sustains the concomitant level of complexity of this brain area in several tasks, such as planning and modulation of movement pathways, cognitive processes involving executive functions, reward and addiction. These possibilities are, of course, not mutually exclusive.
Striatal neurons, including the most abundant medium spiny neurons, receive convergent synaptic modulation from nigral dopaminergic neurons and from cortical glutamatergic projections [38]. The present study showing that lesions of nigral dopaminergic neurons do not significantly affect purinergic receptors present on axons of striatum white matter, but do generate a significant overall decrease in P2X and P2Y receptor proteins from striatal spiny neurons and GABAergic interneurons, thus confirms and extends the involvement of P2 receptors and extracellular ATP to the cortex-basal ganglia circuit [21]. Since dopaminergic denervation affects not only the nigrostriatal dopaminergic pathway but, as a consequence, the corticostriatal glutamatergic pathway with an increase in glutamatergic transmission [39–41] and extracellular glutamate levels in the striatum [42], the reduced P2 receptor protein expression that we demonstrate in striatum gray matter could thus not only be a direct effect of the nigrostriatal inhibition, but also a cause of de-inhibitory mechanisms occurring in the corticostriatal circuit. In this regard, it is common knowledge that extracellular ATP participates in excitatory neurotransmission in the CNS [43], that release of extracellular ATP occurs in CNS under both normal and pathological conditions [44] and, not least, that glutamate release is induced by extracellular ATP in CNS glutamatergic neurons [45].
Neurons of the pars compacta responsible for dopamine production in the brain, which we have shown here to completely lose their array of P2 receptors as a consequence of neurodegeneration induced by 6-OHDA treatment, receive inhibiting signals also from neurons of the pars reticulata that produce GABA [46]. Loss of dopamine neurons in the SNC, one of the main pathological features of Parkinson’s disease leading to a marked reduction in dopamine function in the brain, thus also impedes the inhibitory pathway of SNR, with a consequent overactivation of GABAergic neurons. Our findings that specific expression of both ionotropic P2X1,3,4,6 receptors on GABAergic neurons and metabotropic P2Y4 receptors on astrocytes is remarkably increased in SNR after dopamine denervation thus probably reflects a parallel compensatory overreaction of GABAergic neurons to dopamine shortage. One possible explanation is that purinergic mechanisms might thus play a crucial role in the fine-tuned regulation not only of dopaminergic and glutamatergic cross-talk in striatum, as it occurs in nucleus accumbens [47], but also of GABAergic and dopaminergic interplay in SN, as it occurs in the mesolimbic neuronal circuit [48]. This is consistent with the overall versatile functions accomplished by P2 receptors in the CNS under both normal and pathological conditions [43, 44, 49] and, in particular, with the intermediary role in oligodendrocyte-to-neuron [26], Bergmann glia-to-neuron, and neuron-to-neuron communication [50] proposed for P2 receptors in various brain regions.
In summary, the importance of our work is twofold. We first provide the complete topographical analysis of all known P2X and P2Y receptor subtypes expressed in vivo at their protein levels in rat striatum and SN, which, when considered alongside functional studies, supports a key role for extracellular ATP as a cotransmitter/neuromodulator in these brain areas. Then, we prove that dopamine denervation in the 6-OHDA animal model of Parkinson’s disease generates a significant rearrangement of P2 receptor proteins in these nuclei, therefore disclosing the participation of P2 receptors in the lesioned nigro-striatal circuit. While requiring further investigation, our findings indicate a potential but noteworthy pharmacological and therapeutic novel outcome for Parkinson’s disease.

rat brain
tyrosine hydroxylase
6-hydroxydopamine
parkinson’s disease
purinergic receptors
γ-aminobutyric acid


Best practice guidelines for the molecular genetic diagnosis of maturity-onset diabetes of the young

Aims/hypothesis Mutations in the GCK and HNF1A genes are the most common cause of the monogenic forms of diabetes known as ‘maturity-onset diabetes of the young’. GCK encodes the glucokinase enzyme, which acts as the pancreatic glucose sensor, and mutations result in stable, mild fasting hyperglycaemia. A progressive insulin secretory defect is seen in patients with mutations in the HNF1A and HNF4A genes encoding the transcription factors hepatocyte nuclear factor-1 alpha and -4 alpha. A molecular genetic diagnosis often changes management, since patients with GCK mutations rarely require pharmacological treatment and HNF1A/4A mutation carriers are sensitive to sulfonylureas. These monogenic forms of diabetes are often misdiagnosed as type 1 or 2 diabetes. Best practice guidelines for genetic testing were developed to guide testing and reporting of results.
Introduction
Maturity-onset diabetes of the young (MODY) describes the dominantly inherited disorder of non-insulin-dependent diabetes typically diagnosed before 25 years that was first recognised by Tattersall [1, 2]. MODY is the most common form of monogenic diabetes, accounting for an estimated 1–2% of diabetes in Europe [3, 4], but is often misdiagnosed as type 1 or type 2 diabetes.
The term MODY is used to describe a group of clinically heterogeneous, often non-insulin-dependent forms of diabetes that are defined at the molecular genetics level by mutations in different genes. All show dominant inheritance and are disorders of beta cell dysfunction, but variable features include the age at onset, severity of the hyperglycaemia (and hence risk of complications) and associated clinical features. The most recent classification of diabetes by the American Diabetes Association and the World Health Organization recognises these discrete subtypes of MODY [5].
Mutations in the GCK and HNF1A genes are the most frequent cause of MODY in all populations studied. They account for approximately 70% of cases (see Table 1). The ratio of GCK to HNF1A mutations varies between countries because of different recruitment strategies for genetic testing; blood glucose screening in young, asymptomatic individuals will identify a higher proportion of GCK mutations.
Table 1Genes in which mutations cause MODY Gene symbol (other symbol)GCKHNF1A (TCF1)HNF4APDX1 (IPF1)NEUROD1HNF1B (TCF2)ProteinGlucokinaseHepatocyte nuclear factor-1 alphaHepatocyte nuclear factor-4 alphaInsulin promoter factor-1Neurogenic differentiation 1Hepatocyte nuclear factor-1 betaChromosome locus7p1312q24.3120q13.1213q12.22q31.317q12Gene Accession no.NM_000162.2NM_000545.4NM_000457.3aNM_000209.2NM_002500.2NM_000458.1OMIM * (Gene)138079142410600281600733601724189907OMIM # (Phenotype)125851600496125850606392606394137920Mutation frequency (%) (not known in ~20% of cases)20–5020–50~5<1<1~5The Genbank reference sequence NM_000457.3 refers to the full length P1 transcript, which uses exons 1a and 1b, and not 1d. For the full length P2 transcript, which includes exon 1d, there is a human mRNA sequence AY680697 but no refseq. The convention is to use NM_000457.3 for exons 1a/1b and 2–10, with AY680697 for exon 1d only. HNF4A mutation descriptions in the literature use the translation start codon reported by Chartier et al. in 1994 [45]. However, an alternative start codon nine amino acids upstream was proposed in 1996 [46] and this alternative start codon is used in NM_000457.3. Hence the convention is to report HNF4A mutations using the amino acid methionine at codon 10 in NM_000457.3 as the start codon, with the A of this codon as the first nucleotideaNomenclature for the HNF4A gene is complicated because the gene encodes nine isoforms expressed from two promoters. The liver-specific P1 promoter drives the expression of transcripts 1–3, which include exons 1a and 2–10, and transcripts 4–6, which include exons 1a, 1b, 1c and 2–10. Transcripts 7–9 are expressed from the pancreatic (P2) promoter located approximately 46 kb upstream of the HNF4A transcription start site and exhibit splicing of the upstream exon 1d to exon 2, without the inclusion of sequences from either exons 1a, 1b or 1c
Heterozygous loss-of-function GCK mutations result in mild, stable hyperglycaemia from birth. Microvascular complications are rare, reflecting the fact that HbA1c is normally just above the upper limit of the normal range. Treatment with oral hypoglycaemic agents or insulin is not needed because it rarely changes HbA1c [6]. A genetic diagnosis is important for the small number of children misdiagnosed with type 1 diabetes and treated with insulin [7]. The identification of GCK mutations in women with gestational diabetes can be useful for obstetric management, since their babies who do not inherit the mutation are at risk of macrosomia [8], and it can guide follow-up in the mothers.
Transcription factor mutations in the HNF1A or HNF4A genes cause a similar progressive diabetic phenotype although the penetrance of HNF4A mutations is lower (S. Ellard and A. T. Hattersley, unpublished data). Sensitivity to sulfonylureas means that some patients can transfer from insulin to oral agents [9, 10]. A low renal threshold for glucose is a feature of HNF1A mutations [11] and may provide a useful method of screening at-risk family members during childhood [12].
Mutations identified in the GCK, HNF1A and HNF4A genes include missense, nonsense, splicing, small deletions/insertions/duplications, and splice site and promoter region mutations [13, 14]. Partial and whole deletions have recently been reported in HNF1A and GCK [15]. The location of mutations within the HNF1A gene influences the age at diagnosis; the average age at diagnosis for patients with exon 1–6 mutations that affect all three HNF1A isoforms is younger than for those with mutations in exons 8–10 that affect only isoform HNF1A(A) [16, 17].
Rarer forms of MODY include heterozygous mutations in PDX1 (also known as IPF1; [18, 19]) and NEUROD1 [20, 21], but analysis of these genes is not usually included in routine molecular genetic testing for MODY. Dominantly inherited syndromic forms of diabetes may also be described as MODY subtypes. The renal cysts and diabetes syndrome results from HNF1B mutations, and other features include renal abnormalities, female genital malformations, hyperuricaemia, pancreatic atrophy and abnormal liver function tests [22–24]. Mutations in the CEL variable number tandem repeat cause a syndrome of diabetes and pancreatic exocrine dysfunction [25]. These syndromes and maternally inherited diabetes and deafness caused by the mitochondrial m.3243A→G mutation are not included in these guidelines since testing is guided by the non-endocrine pancreatic or extra-pancreatic clinical features.
A molecular genetic diagnosis of a GCK, HNF1A or HNF4A mutation is important because it confirms a diagnosis of MODY, classifies the subtype, predicts the likely clinical course and may change the patient’s treatment. First-degree relatives will be at 50% risk of inheriting the mutation and asymptomatic individuals may be offered predictive genetic testing (after appropriate genetic counselling) in order to provide reassurance (for those shown not to carry the mutation) or regular blood glucose monitoring with early diagnosis and appropriate treatment (for mutation carriers).
Methods
A group of European clinicians and scientists met on 22 May 2007 at a workshop to formulate best practice guidelines for molecular genetic testing in MODY. Discussions focused on clinical criteria for selection of patients for testing, methodologies, interpretation of results and reporting those results to the referring clinicians.
A draft document was posted on 24 August 2007 and an online editing tool was used by participants to produce consensus guidelines.
Results
Clinical criteria for testing
Mild fasting hyperglycaemia: testing for GCK mutations The finding of raised fasting blood glucose in the range of 5.5–8 mmol/l is unusual in children and young adults. This always raises concern that they may be about to develop type 1 diabetes or the patient has type 2 diabetes. However, a considerable proportion of young, non-obese patients with persistent mild fasting hyperglycaemia will have a heterozygous mutation in the GCK gene. In a cohort of 82 children with incidental hyperglycaemia, 43% had GCK mutations [26]. The phenotype associated with GCK mutations is remarkably similar for all mutations. The following features suggest a diagnosis of a GCK mutation:
The fasting hyperglycaemia is ≥5.5 mmol/l (98% patients), persistent (at least three separate occasions) and stable over a period of months or years [27].HbA1c is typically just above the upper limit of normal and rarely exceeds 7.5%.In an OGTT the increment [(2 h glucose) − (fasting glucose)] is small (71% of patients in the large European study reported by Stride et al. [27] had an increment <3 mmol/l). An increment of 4.6 mmol/l is often used to prioritise testing and corresponds to the 90th centile (S. Ellard and A. T. Hattersley, unpublished data).Parents may have ‘type 2 diabetes’ with no complications or may not be diabetic. On testing, one parent will usually have a mildly raised fasting blood glucose (range of 5.5–8 mmol/l) unless the mutation has arisen de novo. Testing of apparently unaffected parents’ fasting glucose is important when considering a diagnosis of a glucokinase mutation.
Gestational diabetes: testing for GCK mutations Mutations of GCK cause mild fasting hyperglycaemia throughout life and this is often diagnosed during pregnancy when routine testing is performed. Since these patients have consistently raised fasting blood glucose levels, their babies who do not inherit the mutation may be macrosomic [28]. The diagnosis of a GCK mutation is important, not only as the child may subsequently be picked up as having a raised fasting blood glucose and this may lead to concern about type 1 diabetes, but also because the guidelines given to the mother are different from the normal ‘pre-type 2’ diabetic phenotype as they will not deteriorate with time. The following criteria identify when GCK testing is appropriate [29]:
Persistently raised fasting blood glucose in the range of 5.5–8 mmol/l before, during and after pregnancy.An increment of <4.6 mmol/l on at least one OGTT (either during or after pregnancy).A parent may have mild type 2 diabetes but often this has not been detected and so the absence of family history should not exclude the diagnosis.
Children and young adults with diabetes and a strong family history of diabetes: testing for HNF1A mutations The possibility of monogenic diabetes should be considered whenever a parent has diabetes even if they are thought to have type 1 or type 2 diabetes. The most common form of MODY is caused by HNF1A mutations. The clinical characteristics of patients with HNF1A mutations include:
Young-onset diabetes (typically before 25 years old in at least one family member).Non-insulin-dependent outside the normal honeymoon period (3 years), e.g. not developing ketoacidosis in the absence of insulin, good glycaemic control on less than the usual replacement dose of insulin, or detectable C-peptide measured when on insulin with glucose >8 mmol/l.Family history of diabetes (at least two generations). This may be insulin treated and considered to be ‘type 1’ diabetes or ‘type 2’ diabetes. At least two individuals within the family would typically be diagnosed in their 20s or 30s. There may also be an affected grandparent, although often these are diagnosed after 45 years. OGTTs in early stages tend to show a very large glucose increment, usually >5 mmol/l [27]. Some individuals may have a normal fasting level but a value within the diabetic range at 2 h.The absence of pancreatic islet autoantibodies.Glycosuria at blood glucose levels <10 mmol/l is often seen, as these patients have a low renal threshold [11].Marked sensitivity to sulfonylureas resulting in hypoglycaemia despite poor glycaemic control before starting sulfonylureas [9, 30].Several features suggesting monogenic diabetes rather than a diagnosis of young-onset type 2 diabetes should be considered: no marked obesity or evidence of insulin resistance in diabetic family members, absence of acanthosis nigricans and whether the family is from an ethnic background with a low prevalence of type 2 diabetes (e.g. of European descent).
Children and young adults with diabetes and a strong family history of diabetes: testing for HNF4A mutations Diabetes caused by mutations in the HNF4A gene is considerably less common (Table 1) than HNF1A mutations. The clinical characteristics are similar, except there is not a low renal threshold and the age of diagnosis may be later [31]. HNF4A mutations should be considered when HNF1A analysis does not detect a mutation but the clinical features are strongly suggestive of HNF1A. Patients are often sensitive to sulfonylureas [32]. HNF4A mutations are associated with macrosomia (approximately 56% of mutation carriers) and transient neonatal hypoglycaemia (approximately 15% of mutation carriers) [33, 34]. The possibility of HNF4A mutations should be considered when diabetic family members have marked macrosomia (>4.4 kg at term) or if diazoxide-responsive neonatal hyperinsulinism has been diagnosed in the context of familial diabetes.
Babies with diazoxide-responsive neonatal hyperinsulinaemic hypoglycaemia and a strong family history of diabetes: testing for HNF4A mutations Mutations of HNF4A are a cause of neonatal hypoglycaemia that remits during infancy or early childhood, with diabetes developing later in life [34]. Macrosomic babies with diazoxide-responsive hyperinsulinism and a strong family history of diabetes (see characteristic 3 in the section above entitled Children and young adults with diabetes and a strong family history of diabetes: testing for HNF1A mutations) should be considered for HNF4A mutation screening.
Testing methodology
The mutation screening methodology should be described in the report [e.g. sequencing, denaturing high-performance liquid chromatography (dHPLC), conformation-sensitive capillary electrophoresis (CSCE)] together with the sensitivity. PCR primers should be checked for primer binding site single nucleotide polymorphisms (SNPs; a useful tool is available at http://ngrl.man.ac.uk/SNPCheck/index.html). Gene dosage analysis may be useful if a diagnosis of MODY is strongly suspected and no mutation is found on mutation screening.
Interpretation of results
The textbox includes recommended interpretations for the most common reporting scenarios.
Reporting
Each laboratory has its own reporting format and general guidance on reporting is available from the European Molecular Genetics Quality Network (http://www.emqn.org), the UK Clinical Molecular Genetics Society (http://www.cmgs.org) and the Swiss Society of Medical Genetics (http://www.ssgm.ch). A one page report is the preferred format.
The report should state the methodology and specify the gene, exons and/or mutations tested for. If promoter sequences are examined then the report should specify the nucleotides analysed. An estimation of the assay sensitivity is particularly useful for pre-screening techniques such as dHPLC, CSCE etc. The use of mutation nomenclature approved by the Human Genome Variation Society (http://www.hgvs.org/mutnomen) is strongly recommended. The gene accession number (with version) is required in order to describe mutations unambiguously (see Table 1). The A nucleotide of the ATG start codon is numbered +1.
Reports describing novel variants should state that the variant is novel and include the evidence in support of pathogenicity. This might include the absence from a large series of ethnically matched controls or MODY patients (testing of 210 normal chromosomes is necessary to achieve at least 80% power to detect a polymorphism present in 1% of the population [35]). Testing of other affected relatives is recommended in order to check for co-segregation and to calculate the LOD score in suitable sized pedigrees (LOD scores of ≥1 or ≥3 are suggestive or conclusive of linkage, respectively).
For missense variants the evidence for pathogenicity might include conservation across species and a significant amino acid substitution. Several programs are available that predict the pathogenicity of a missense variant based upon amino acid conservation (SIFT; http://www.blocks.fhcrc.org/sift/SIFT.html) or the structure and function of the protein (PolyPHEN; http://www.genetics.bwh.harvard.edu/pph) but they should be used to supplement other pieces of evidence rather than in isolation.
Both missense and silent variants can affect splicing if the mutation is within an exon splicing enhancer or exon splicing silencer. Splice predictor software programs (http://www.fruitfly.org or http://rulai.cshl.edu/cgi-bin/tools/ESE3/esefinder.cgi?process=home) may aid interpretation. Base substitutions affecting the conserved splice donor (GT) site, splice acceptor (AG) site or the conserved A nucleotide within the branch site are highly likely to be pathogenic but splice predictor software may be useful in the interpretation of other intronic variants. Analysis of patient mRNA is often informative but lymphoblastoid cell lines are usually required because of the low levels of expression of the MODY genes in blood. Sequence analysis of RT-PCR products amplified from lymphoblastoid cell mRNA has demonstrated exon skipping, retention and the use of cryptic splice sites for a variety of intronic mutations in the GCK, HNF1A and HNF1B genes [36–38].
Novel promoter variants may be investigated by examination of known transcription binding sites or by in vitro transfection experiments [39–42]. They may also alter mRNA expression levels, which may be measured by allele-specific real-time PCR [43].
Polymorphisms
Some laboratories include details of polymorphisms detected in the report. The reasons for doing this include: (1) making all data available to the requesting clinician based on the rationale that a polymorphism may later be reclassified as a mutation; and (2) identifying heterozygous SNPs excludes a gene deletion involving the exon(s) in question.
However, this information can cause confusion or even misinterpretation of the result (A. T. Hattersley, unpublished data) and it certainly adds to the length of the report. While in some cases there may be reports in the literature of an association with type 2 diabetes or reduced insulin secretion, these polymorphisms do not cause MODY and we recommend that they should be excluded from the report.
Treatment
Individual treatment recommendations are outside the jurisdiction of a molecular genetics report since this is the referring clinician’s responsibility. It is useful to include an appropriate reference if there is evidence in the literature for a particular treatment (e.g. low-dose sulfonylureas in HNF1A/4A MODY) associated with the genetic diagnosis.
Other issues
Genetic counselling should be provided for all asymptomatic individuals requesting predictive testing. We recommend that unaffected relatives are offered a biochemical test first (fasting blood glucose for GCK mutations or OGTT for HNF1A/HNF4A mutations). If the biochemical test is consistent with a diagnosis of diabetes or hyperglycaemia then the genetic test will be diagnostic, not predictive.
For families requesting predictive testing for children too young to provide informed consent, referral to a specialist clinical genetics unit (or equivalent) is strongly recommended. Reasons for testing children include (1) to remove the uncertainty around the child’s status, and (2) to assist with management, as a negative test would mean that monitoring of blood glucose/glycosuria would not be necessary [44].
Conclusions
Molecular genetic testing is useful in patients with MODY because it confirms a diagnosis of monogenic diabetes, predicts likely clinical course, defines risk for relatives and determines treatment.
At the present time, molecular genetic testing for MODY is relatively expensive and phenotypic selection prior to testing is normal practice. With the development of new technologies it is likely that these costs will decrease in time and that the analysis of genes associated with monogenic diabetes may become routine for all newly diagnosed patients. In the meantime we hope that these guidelines will be useful in determining which patients should be offered testing, and in the interpretation and reporting of the test results.
Below is the link to the electronic supplementary material.
ESM
(PDF 12.9 kb)

best practice
maturity-onset diabetes of the young
gck
hnf1a
hnf4a
mody
monogenic diabetes


Short-term beneficial effects of methylene blue on kidney damage in septic shock patients

Objective We previously demonstrated that upregulation of renal inducible nitric oxide synthase (iNOS) is associated with proximal tubule injury during systemic inflammation in humans. In this study we investigated the short-term effect of methylene blue (MB), an inhibitor of the NO pathway, on kidney damage and function in septic shock patients.
Introduction
Refractory septic shock remains the major cause of death in noncoronary intensive care units, especially when accompanied by multiple organ failure, with an estimated mortality rate of 50–60% [1]. The incidence of acute renal failure in refractory septic shock is approx. 40–50% [2]. Nitric oxide has emerged as an important contributory factor to the pathogenesis of septic shock. We previously demonstrated that induction of renal NO is associated with proximal tubule injury during systemic inflammation in humans [3]. NO stimulates soluble guanylate cyclase (sGC) by binding to its heme moiety, which generates cyclic guanosine monophosphate (cGMP) [4]. In the kidney NO and cGMP production are associated with lipopolysaccharide-induced renal proximal tubular cell toxicity [5]. Selective sGC inhibition during septic shock in rats resulted in an attenuation of renal dysfunction [6], indicating that blocking sGC may be a potential therapeutic strategy to treat septic shock-associated renal failure.
Methylene blue (MB) binds to sGC, blocks cGMP production and has the ability to scavenge NO and to inhibit NO synthases [7, 8]. Although several controlled and uncontrolled clinical studies showed beneficial effects of MB on the hemodynamic instability during septic shock (reviewed in [9]), there are no studies on the putative protective renal effects. We examined the short-term effects of continuous infusion of low-dose MB (1 mg/kg per hour) in patients with septic shock on urinary excretion of the acute kidney injury markers, cytosolic glutathione S-transferases (GSTs) present in proximal tubule (GSTA1-1) and distal tubule (GSTP1-1) [10].
Material and methods
Patients
Nine patients received a 4 h continuous infusion of 1 mg/kg per hour MB (1% w/v) provided by the VieCuri Medical Center pharmacy (for inclusion criteria see Electronic Supplementary Material, ESM). Arterial blood and catheterized urine were collected at several time points during the first 24 h. Clinical parameters, the severity of illness using Acute Physiology and Chronic Health Evaluation II (APACHE II), and Sepsis-Related Organ Failure Assessment (SOFA) were recorded.
Chemical assays
Biochemical parameters were determined by routine clinical chemistry. Hemoglobin, methemoglobin, and bilirubin were measured to assess possible side effects of MB, such as hemolytic anemia and methemoglobinemia [11–13]. Total amount of the stable NO metabolites, nitrate and nitrite, measure of NO radicals production, and the amounts of GSTA1-1 and GSTP1-1 in urine were assayed as described [3]. The blue color in urine due to MB excretion did not affect the chemical assays.
Statistical analysis
Values are given as mean ± S.E. or as median (25–75% range) depending on their distribution. Differences between experimental groups were tested by analysis of variances for repeated measures. A two-tailed p-value less than 0.05 was considered statistically significant.
Results
Patients
Seven patients ultimately died in the intensive care unit, one of refractory shock (within 12 h) and six patients because of multiple organ failure. In the latter group two died within 7 days and the remaining four within 28 days after intervention. The mean calculated predicted mortality rate was 61%, and all patients had at least three organ failures, reflecting a mean SOFA score of 11.1 ± 0.9. The median stay at the intensive care unit was 16 days (range 7–24); the two survivors stayed 89 days (52–121) in hospital. Pathogenic organisms isolated by culture and site of infection are illustrated in the ESM. Median C-reactive protein was 178 mg/l (118–189); all patients had lactic acidemia (median 2.7 mmol/l, range 2.1–3.7) and thrombocytopenia (68 × 109/l, 50–104). The median MAP increased slightly by 5 mmHg (2–11) from 69 (65–70) at baseline to 74 (68–82) 3 h after the start of MB (p < 0.05) with no change in norepinephrine infusion rate. Methemoglobinemia or hemolytic anemia did not develop after MB (data not shown). All MB-treated patients showed blue-coloring of urine and skin.
MB attenuates NO formation
The concentration NO metabolites in plasma was higher in septic shock patients than in to healthy volunteers [3] but did not change after MB administration (Fig. 1a). In contrast, MB significantly attenuated the urinary excretion of NO metabolites by a median 90% (75–95%, p < 0.05) from baseline (233 μmol/mmol creatinine, 112–536) to 6 h (37, 10–87) after the start of MB (Fig. 1b). At 4h the MB infusion was stopped, after which the median excretion of urinary NO metabolites increased by 135% (65–795%, p < 0.05) within 6–24 h after MB treatment (Fig. 1b).
Fig. 1NO metabolites in plasma and urine and the urinary excretion of tubular injury markers glutathione S-transferase (GST) A1-1 and P1-1. NO metabolite levels in plasma (a, n = 9) and urine (b, n = 8) and levels of GSTA1-1 (c, proximal tubule, n = 8) and GSTP1-1 (d, distal tubule, n = 8) were measured in urine at various times after MB administration in septic shock patients. The urinary excretion of NO metabolites and GSTs was corrected for creatinine excretion. Data are expressed as median with 25–75% range and analyzed by analysis of variance with repeated measures over the two time periods. *p < 0.05 vs. baseline, # p < 0.05 vs. 6 h after MB treatment
MB attenuates kidney damage
All patients showed impaired renal function with oliguria and mild proteinuria (Table 1). One patient suffered from anuria during the first day. Six patients required continuous venovenous hemofiltration renal replacement therapy with a flow of 35 ml/kg (for median 8.5 days, 4–13), of which 3 during the MB infusion. The creatinine clearance improved by median 51% (18–173%, p < 0.05) during the first 24 h after MB but was still strongly impaired (Table 1). The urinary excretion of both GSTA1-1 and GSTP1-1 was elevated in all septic shock patients, indicating both proximal and distal renal tubule damage. During the first 6 h of MB urinary excretion of GSTA1-1 and GSTP1-1 was attenuated by median 45% (10–70%) and 70% (40–85%) vs. baseline (Fig. 1c, d, p < 0.05). After ending MB infusion urinary excretion of GSTA1-1 and GSTP1-1 increased again, although not significantly (Fig. 1c, d).
Table 1Kidney function parameters of septic shock patients (n = 8) treated with MBMedianRangeTotal urine volume, 0–24 h (ml)495169–885Protein excretion, 0–24 h (mg/day)342245–434Creatinine clearance (ml/min) Baseline 8.2 4.2–17.4 24 h 10.6* 9.6–14.8Blood urea nitrogen Baseline 17.8 10.8–20.0 24 h 17.3 10.4–22.5Fractional excretion of sodium (%) > 2%  Baseline (n = 0)––  24 h (n = 3) 3.1 2.6–3.6 < 1%  Baseline (n = 8) 0.6 0.3–0.7  24 h (n = 5) 0.4 0.2–0.5p < 0.05 vs. baseline
Discussion
Several clinical studies in septic shock patients have investigated the effects of MB on the heart, vascular wall, and lungs [9]. This is the first report demonstrating that MB attenuates kidney damage in human septic shock. To determine the effect of NO pathway inhibition on renal damage we examined the urinary excretion of early tubular injury markers and found that MB inhibited the NO pathway and preserved the integrity of renal tubules. After termination of MB infusion these parameters returned to their elevated pretreatment levels. Both plasma concentrations (more than twofold) and urinary NO metabolites levels in our septic shock patients were much higher than in healthy volunteers, as demonstrated earlier [3]. Hydrocortisone may have inhibited iNOS activation, however, the patients received a continuous infusion of hydrocortisone that started before MB infusion and continued during the 24 h period. Therefore the observation that urinary NO metabolite excretion was attenuated only in the first 6 h after the start of MB suggests that this effect is not related to steroids. Although urinary NO metabolite excretion was attenuated, we did not find a reduction in plasma NO metabolites. This is in contrast with an earlier report, in which patients received a bolus injection of MB prior to the continuous infusion [14].
Renal failure in septic shock patients is a complex and multifactorial disease process. During septic shock the systemic vasodilation increases renal sympathetic activity and angiotensin concentration which results in intrarenal vasoconstriction with sodium and water retention and decreased glomerular filtration rate [2]. We previously demonstrated that induction of renal iNOS, constitutively expressed in the kidney [15], is associated with proximal tubule injury during systemic inflammation in humans [3]. As a result of its active secretory transport function and role in urine concentration, the proximal tubule is a susceptible target and often the first site of damage [16]. Therefore inhibition of peroxynitrite formation from excessively produced NO and superoxide [17] by MB may be beneficial for the kidney during septic shock, possibly explained by the local accumulation of MB in renal proximal tubules [18]. Global hemodynamic variables can influence renal function, however, only a small but statistically significant increase was found for mean arterial pressure, whereas other global hemodynamic parameters did not change during MB treatment.
Since the detailed nature of our investigation, obviously these methods are not feasible in a large-scale clinical intervention study. The most elegant way to examine the effects of MB would be in a randomized, placebo-controlled cross-over study. However, with such critically ill patients this design may be considered unethical. We deliberately chose a subgroup of severe septic shock patients with a high chance of sepsis-induced renal damage for two reasons: first, this is the group of patients in which MB is used as a “last resort therapy” and, second, to demonstrate the putative beneficial effects of MB on renal damage. With an estimated standard deviation of 36% in urinary GST excretion, 80 patients would be needed to demonstrate with 80% power a 10% reduction in renal injury. This number of patients was not feasible for our investigations. Therefore we decided to determine the parameters before, during and after MB infusion during a 24 h period, which allows each patient to serve as own control. Because of the observational nature and limited size of the present study and the heterogeneity of the patient population, our findings warrant conformation on hard endpoints by a larger clinical trial. However, in our view, first a long-term study is necessary to assess the safety of chronic MB administration in septic patients with refractory shock. Promising effects of MB were found in a trial with vasoplegic patients after cardiac surgery treated with MB [19], in which a reduction in both mortality and incidence of renal failure was observed.
In conclusion, short-term infusion of MB in septic patients with refractory shock, is associated with a decrease in NO production and an attenuation of the urinary excretion of renal tubular injury markers.
Electronic supplementary material
Electronic Supplementary Material (DOC 26K)
Electronic Supplementary Material (DOC 37K)

inducible nitric oxide synthase expression


Involvement of P2X and P2Y receptors in microglial activation in vivo

Microglial cells are the primary immune effector cells in the brain. Extracellular ATP, e.g., released after brain injury, may initiate microglial activation via stimulation of purinergic receptors. In the rat nucleus accumbens (NAc), the involvement of P2X and P2Y receptors in the generation of microglial reaction in vivo was investigated. A stab wound in the NAc increased immunoreactivity (IR) for P2X1,2,4,7 and P2Y1,2,4,6,12 receptors on microglial cells when visualized with confocal laser scanning microscopy. A prominent immunolabeling of P2X7 receptors with antibodies directed against the ecto- or endodomain was found on Griffonia simplicifolia isolectin-B4-positive cells. Additionally, the P2X7 receptor was colocalized with active caspase 3 but not with the anti-apoptotic marker pAkt. Four days after local application of the agonists α,βmeATP, ADPβS, 2MeSATP, and BzATP, an increase in OX 42- and G. simplicifolia isolectin-IR was observed around the stab wound, quantified both densitometrically and by counting the number of ramified and activated microglial cells, whereas UTPγS appeared to be ineffective. The P2 receptor antagonists PPADS and BBG decreased the injury-induced increase of these IRs when given alone and in addition inhibited the agonist effects. Further, the intra-accumbally applied P2X7 receptor agonist BzATP induced an increase in the number of caspase-3-positive cells. These results indicate that ATP, acting via different P2X and P2Y receptors, is a signaling molecule in microglial cell activation after injury in vivo. The up-regulation of P2X7-IR after injury suggests that this receptor is involved in apoptotic rather than proliferative effects.
Introduction
Microglial cells are the principal intrinsic immune competent cells of the brain. Resting microglial cells with a ramified morphology become activated under almost all pathological conditions, e.g., mechanical or chemical injury or inflammation. They change into ameboid-shaped motile phagocytotic and cytotoxic cells, which invade the site of injury and initiate repair or defense processes by secreting proteases, neurotrophic factors, and inflammatory mediators [1, 2]. This suggests that microglial cells are able to sense signal molecules entering the extracellular space by disturbances of brain homeostasis and promoting their transformation into the active state [3, 4]. Further, microglia express major histocompatibility complex antigen class II and may perform phagocytotic activity to clear cellular debris [4–7].
One of the most prominent compounds released by injury is the purine nucleotide ATP. Extracellularly applied ATP induces the activation of a cation conductance and a subsequent increase in intracellular Ca2+ [8]. ATP was shown to stimulate microglial cells to respond with rapid movements of their processes [9]. Further, ATP also induced the outgrowth of processes and membrane ruffling in cultured systems [10, 11]. Dependent on the concentration of ATP applied to in vitro systems, microglial cells secrete various biologically active substances, such as the cytokines tumor necrosis factor-α (TNF-α) [12], interleukin (IL)-1β [13, 14], or IL-6 [15]. Boucsein and colleagues [16] have shown that in the presence of purinergic ligands, the lipopolysaccharide-induced release of TNF-α, IL-6, IL-12, and macrophage inflammatory protein-1α was attenuated. In patch-clamp investigations with ATP-induced Ca2+ wave recordings of astrocytes, a current response in an adjacent microglial cell was observed, suggesting that microglia can sense the activity of astrocytes [17].
All these data suggest that a variety of ATP-sensitive ionotropic (P2X) and metabotropic (P2Y) receptors is expressed at resting and/or activated microglial cells [18–22]. The functional expression of ATP receptors on cultured microglia is well known [18, 23, 24]. The basic problem in studying this type of glial cells is that any manipulation ex vivo or in vitro will promote their transformation into the active form. We used an in vivo model of a stab wound injury in the rat nucleus accumbens (NAc) to observe the P2 receptor-mediated response of microglial cells to ATP released by injury. This technique enables local administration of receptor-specific purinergic ligands by microinfusion. Furthermore, our investigations focused especially on the P2X7 receptor subtype on microglial cells in vivo and the functional consequences of injury-induced stimulation. The possible involvement of P2X7 receptors in apoptotic and proliferative processes in microglia and astrocytes, respectively, was also investigated.
Material and methods
Materials
Ketamine hydrochloride (Ketanest; Ratiopharm, Ulm, Germany), xylazine hydrochloride (Rompun; Bayer, Leverkusen, Germany), and thiopental natrium (Trapanal, Altana Pharma, Konstanz, Germany) were used for anesthesia. For intra-accumbal microinjection, 2-methylthioATP (2MeSATP), α,β-methyleneATP (α,βmeATP, both RBI; Natick, MA, USA); adenosine 5′-(β-thio)diphosphate (ADPβS), 2′,3′-O-(4-benzoyl-benzoyl)ATP (BzATP), brilliant blue G (BBG), and 5-bromo-2′-deoxyuridine (BrdU) (all from Sigma, Deisenhofen, Germany); uridine 5′-O-(γ-thio)triphosphate (UTPγS, Inspire Pharmaceuticals Inc., Durham, NC, USA); pyridoxal-5′-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS; Biotrend, Köln, Germany); and artificial cerebrospinal fluid [ACSF (in mM): 126 NaCl; 2.5 KCl; 1.2 NaH2PO4; 1.3 MgCl2, and 2.4 CaCl2; pH 7.4] were used.
Antibodies directed against glial fibrillary acidic protein (GFAP; mouse anti-GFAP, Sigma, Deisenhofen, Germany; rabbit anti-cow GFAP, DakoCytomation, Hamburg, Germany); BrdU (mouse anti-BrdU, Clone Bu20a; DakoCytomation), active caspase 3 (rabbit, Promega, Madison, WI, USA); OX 42 (CD 11b, mouse anti-rat, complement receptor type 3; Serotec, Oxford, UK); (a) lectin from Bandeiraea simplicifolia (Griffonia simplicifolia) isolectin B4 (BSI-B4, peroxidase labeled) and (b) lectin from B. simplicifolia (G. simplicifolia) isolectin B4 (GSA-B4, biotin conjugated; Sigma, Deisenhofen, Germany, each); phosphorylated serine/threonine kinases (pAkt1/2/3; rabbit, Ser 473, raised against the short amino acid sequence containing phosphorylated Ser 473 of Akt 1, Akt 2, and Akt 3 of mouse origin, recommended for the detection of Ser 473 phosphorylated Akt 1 and corresponding phosphorylated Akt 2 and Akt 3 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); and the following P2 receptor antisera were used: rabbit anti-P2X1, anti-P2X2, and anti-P2X4,5,6 (Alomone Labs, Jerusalem, Israel) as well as rabbit anti-P2X7 receptor-subtype (intracellular C-terminus binding, Alomone Labs), anti-P2X3 (guinea pig; Neuromics Inc., Northfield, MN, USA). Furthermore, a goat anti-rat ecto-P2X7 receptor antibody (a kind gift of Dr. M. Kim; [25]) was used for immunofluorescence double-labeling studies. Additionally, anti-P2Y1, anti-P2Y2, anti-P2Y4, anti-P2Y12 (rabbit; Alomone Labs, Jerusalem, Israel), anti-P2Y6 (rabbit; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-P2Y1 (rabbit, GlaxoSmithKline, Brentford, Middlesex, UK) receptor antibodies were tested. The secondary carbocyanine (Cy)2- and Cy3-conjugated IgGs as well as Cy2- and Cy3-conjugated streptavidin (Jackson ImmunoResearch, West Grove, PA, USA) were used. For histochemical detection, 3,3′-diaminobenzidine hydrochloride (DAB; Sigma Chemical Co., St. Louis, USA) was applied.
Animals
Male Wistar rats (280–320 g) were housed under a 12-h light, 12 h-dark cycle and allowed access to lab food and water ad libitum. All procedures using animals were approved by the committee of Animal Care and Use of the relevant local governmental body in accordance with the law of experimental animal protection.
Surgery/microinjection
Anesthetized rats were fixed in a stereotaxic frame. At the coordinates AP = 1.7 mm (rostral to bregma), L = 1.5 mm (lateral to the sagittal suture), and V = 6.5 mm (below the surface of the hemisphere), the injection cannulae connected to a syringe pump via an FEP tubing was inserted into the NAc [26]. By microinfusion, rats received ACSF, which was used as control as well as vehicle for the following P2 receptor agonists: 2MeSATP (nonselective), α,βmeATP (P2X1,3), ADPβS (P2Y1,11,12,13), UTPγS (P2Y4,6), given as 0.1 nmol each, and BzATP (preferential P2X7) 0.3 nmol. As antagonists, PPADS (30 pmol; nonselective) and BBG (1 pmol, P2X7) were applied. When the effects of the P2 receptor agonists were pharmacologically proven, the microinfusion of the respective antagonists preceded infusion of the agonist and antagonist mixture. As a proliferation marker, BrdU (0.1 nmol) was applied together with the antagonist, the agonist, or ACSF alone. Test substances were injected in a volume of 1 μl at a rate of 12 μl/h.
Immunocytochemical studies and double-immunofluorescence studies
Immunocytochemical and double-immunofluorescence studies were done as previously described [26, 27]. After a postinjection time of 2 h and 4 days, the rats were transcardially perfused under thiopental sodium anesthesia with paraformaldehyde (2%) in sodium acetate buffer (pH 6.5; solution A) followed by paraformaldehyde (2%)/glutaraldehyde (0.1%) in sodium borate buffer (pH 8.5; solution B). The brains were immediately removed and stored overnight in solution B without glutaraldehyde. Serial coronal sections (50-μm thick) from the NAc were obtained using a vibratome (Leica, Typ VT 1000S, Nussloch, Germany) and collected as free-floating slices (0.1 M Tris; pH 7.6).
Immunocytochemical studies
BSI immunoreactivity
Free-floating sections were rinsed with 0.05 M Tris-buffered saline (TBS, pH 7.6) and were treated with 1% hydrogen peroxide for 30 min to inactivate endogenous peroxide activity. Immunolabeling was performed with lectin from BSI-B4 (1:200) in TBS containing 2% bovine serum albumin overnight at 4°C, followed by washing in 0.05 M Tris buffer (pH 8.0). Peroxidase activity was visualized with DAB (0.07%) containing nickel ammonium sulphate (1%) and hydrogen peroxide, which renders a black reaction product.
Active caspase 3 immunoreactivity
Free-floating sections were rinsed with 0.1 M TBS (pH 7.4) and treated with 1% hydrogen peroxide for 25 min to inactivate endogenous peroxide activity. Immunoreactivity (IR) was studied with rabbit anti-active caspase 3 (1:500) in TBS containing 10% normal horse serum (NHS) and 0.1% Triton X-100 overnight at 4°C, followed by biotinylated horse anti-rabbit IgG (1:100, Vector Labs. Burlingame, CA, USA) and preformed streptavidin/biotin-peroxidase complex (1:125, StreptABComplex; DakoCytomation) for 2 h. DAB (0.05%) served as chromogen.
GFAP/BrdU immunoreactivity
The procedure was applied to free-floating slices as previously described [26]. Briefly, the GFAP staining procedure was carried out with rabbit anti-cow GFAP antiserum (1:600) and biotinylated protein A (1:400; Calbiochem, La Jolla, CA, USA). To detect the astroglial marker, the streptavidin/biotin technique based on a StreptABComplex (1:125) and DAB (0.05%) as chromogen were used. Mitotic astrocytes were identified by immunostaining of the incorporated BrdU. After DNA denaturation (2 N HCl) and neutralization (borate buffer; 0.15 M; pH 8.5), the slices were incubated with a mouse monoclonal antibody against BrdU (1:75), followed by incubation with horse biotinylated anti-mouse immunoglobulins (1:100; Vector Labs., Burlingame, CA, USA) and with ABC Elite Kit (1:50; Vectastain; Vector). Peroxidase activity was visualized with DAB (0.07%) containing nickel ammonium sulphate (1%) plus cobalt chloride (1%) (DAB-Ni/Co) and hydrogen peroxide, which renders a black reaction product. For GFAP/BrdU double-staining experiments to characterize mitogenic changes, slices were first processed for anti-GFAP labeling followed by BrdU immunolabeling.
Double-immunofluorescence studies
After washing with TBS (0.05 M; pH 7.6) and blocking with normal goat serum (1% NGS) in TBS, slices were incubated in an antibody mixture of mouse anti-OX 42 (CD11b; 1:100) and rabbit anti-P2X receptor antibodies (P2X1 1:400; P2X2 1:400; P2X4 1:400; P2X5 1:400; P2X6 1:400; P2X7 1:1,000) or P2Y receptor antibodies [P2Y1 1:400; P2Y2 1:1,000; P2Y4 1:1,000; P2Y12 1:400 (Alomone Labs); P2Y1: 1:1,500 (GlaxoSmithKline); P2Y6 1:200 (Santa Cruz Biotechnology, Inc.)] with 0.1% Triton X-100 in 1% NGS in TBS for 48 h at 4°C. The secondary antibodies employed for simultaneous localization of the two primary antisera were Cy2-conjugated goat anti-mouse IgG (1:500) and Cy3-conjugated goat anti-rabbit IgG (1:800), respectively. Cy2-conjugated goat anti-guinea pig IgG (1:400) and Cy3-conjugated goat anti-mouse IgG (1:1,000) were used for visualization of guinea pig anti-P2X3 (1:1,000). Sections were washed three times for 5 min each in 1% NGS [or 5% fetal calf serum (FCS) for guinea pig anti-P2X3] in TBS and then incubated for 2 h in a solution containing a mixture of the secondary antibodies with 1% NGS (5% FCS) in TBS.
GSA-B4 and P2X or P2Y double labeling was performed in two steps: first the incubation of the slices with GSA-B4 (1:300), followed by treatment with Cy2-conjugated streptavidin (1:350); and second, by incubation with rabbit P2X/Y antibodies followed by Cy3-conjugated goat anti-rabbit IgG (1:800) as described above. The ecto-P2X7 receptor antibody (goat anti-rat ecto-P2X7 receptor, 0.12 μg/ml [25]) was combined with rabbit anti-caspase 3 (1:500), rabbit anti-pAkt (1:600), or rabbit anti-P2X7 (C-terminal, 1:1,000) and subsequent detection with Cy2-conjugated donkey anti-goat IgG (1:1,000) and Cy3-donkey anti-rabbit IgG (1:1,000). Incubation of slices with goat anti-rat ecto-P2X7 (1:600), rabbit anti-active caspase 3 (1:500), or rabbit anti-pAkt (1:600) and GSA-B4 (1:300) was followed by treatment with Cy3-conjugated donkey anti-goat (1:1,000) or Cy3-conjugated goat anti-rabbit IgG (1:400) and Cy2-conjugated streptavidin (1:350). Control experiments were carried out without the primary P2 receptor antibody or by preadsorption of the antibody with the peptides used for their generation. When slices were incubated with PBS instead of the primary antibody or with primary antibody serum, which had been preabsorbed with peptide antigen for 1 h before use, no immunofluorescence with either of the control procedures was observed. After intensive washing and mounting on glass slides, all stained sections were dehydrated in a series of graded ethanol, processed through n-butylacetate, coverslipped with entellan (Merck, Darmstadt, Germany), and analyzed by light microscopy or confocal laser scanning microscopy.
Confocal microscopy
Double immunofluorescence was investigated by a scanning confocal microscope (LSM 510, Zeiss, Oberkochen, Germany) equipped with an argon laser emitting at 488 nm (yellow-green Cy2-immunofluorescence) and a helium/neon laser emitting at 543 nm (red Cy3-immunofluorescence).
Quantification and statistical analysis
Changes in BSI-B4-IR were determined by densitometry (in regions 1 and 2, Fig. 1a) using a digital image analyzer (Diana II) in combination with an advanced image data analyzer (AIDA 2.0). Results are expressed as percentage of the BSI-B4-IR of the ACSF-treated sides in the same regions.
Fig. 1a A rat brain slice including needle tracts and regions of interest within the nucleus accumbens (NAc) in which immunoreactivity (IR) and cells were evaluated (1 core 1; 2 core 2; according to [29]). b BSI-B4-marked microglial cells after stab-wound injury (overview). c Quantification of the number of activated microglial cells after injury and microinjection of P2-receptor-agonists 2MeSATP, ADPβS, α,βmeATP (0.1 nmol each), and BzATP (0.3 nmol) after a postinjection time of 4 days. Values are expressed as percentage of ACSF-treated controls and represent mean ± SEM of six animals per group. d–f BSI-B4-IR of microglial cells in the NAc of rats, illustrating changes in the glial morphology: d A great number of resting (process-bearing) microglial cells (arrows) under control conditions; e, f changes in the number of resting microglial cells (arrow) and activated microglial cells (arrowhead) after different treatment conditions (scale bar = 200 μm). g Quantification of the effects of agonists 2MeSATP, ADPβS, α,βmeATP (0.1 nmol each), and BzATP (0.3 nmol) alone and in combination with PPADS (0.1 nmol/0.03 nmol each) or BBG (1 pmol) on BSI-B4-IR of microglial cells in brain slices of the NAc of the rat after a postinjection time of 4 days. Data are expressed as percentage of the ACSF-treated side and represent the mean ± SEM of six animals per group (*p < 0.05, vs. ACSF group; +p < 0.05, agonist vs. antagonist/agonist group)
Additionally, the number of BSI-B4-labeled ramified and activated microglial cells as well as GFAP and GFAP/BrdU double-labeled cells were counted under a light microscope (Axioskop; Zeiss) with a 20x objective within a square (0.5 × 0.5 mm) in region 1 (Fig. 1a). Each value represents five replications for each condition. The results were expressed as a percentage of ACSF-treated sides of the same regions. The individual groups were compared with one-way analysis of variance (ANOVA) using Bonferroni t test. A probability level of 0.05 or less was considered to be statistically significant.
Results
Immunocytochemistry
After mechanical injury (Fig. 1a,b), a characteristic BSI-B4 immunolabeling around the stab wound was observed in comparison with untreated controls, decreasing in relation to the distance from the lesion. In untreated rats, a great number of resting (process-bearing) microglial cells was present (Fig. 1d, arrows), whereas after stab-wound injury, changes in the number of resting microglial cells with retracted processes (Fig. 1e, arrow) and activated microglial cells (Fig. 1f, arrowhead) were observed. Quantification of BSI-B4-immunolabeling in the NAc (region 1; Fig. 1c) of rats 4 days after stab-wound injury indicated a variable influence of P2 receptor agonists on the number of activated cells. Whereas α,βmeATP had no effect, 2MeSATP was more potent in tendency than was ADPβS in increasing the number of activated microglial cells. BzATP, which was injected into the NAc in a higher dose than were the previous ligands [28], also induced a marked enhancement of activated microglial cells (Fig. 1c). UTPγS was without effect, both on the number of activated microglia and on resting microglia (not shown).
Quantification of BSI-B4-IR in the NAc (region 1 plus 2) by densitometry showed a significant increase for all agonists used with the exception of UTPγS (not shown) when compared with ACSF-treated animals. The effects of α,βmeATP, ADPβS, and 2MeSATP could be inhibited by pretreatment with PPADS and those of BzATP by pretreatment with BBG (Fig. 1g). PPADS and BBG given alone inhibited and failed to alter BSI-B4-IR, respectively.
Double-immunofluorescence studies at P2X and P2Y receptor subtypes
Multiple immunofluorescence labeling in combination with laser scanning microscopy was used to characterize the P2X/Y receptor subtype expression after injury in comparison with untreated controls. Under control conditions, no P2X or P2Y (with the exception of P2Y1) receptor-IR was coexpressed on single GSA-B4-positive cells. The mechanical injury associated with the introduction of the injection cannula resulted in the expression of P2X1,2,4,7 and P2Y1,2,4,6,12 receptors in the lesioned area, observed earliest 2 h until 4 days after injury. An up-regulation of immunolabeling of P2X1,2,4,7 receptor subtypes was unequivocal; examples are shown in Fig. 2 (a–i). In the affected area, P2Y1,2 and P2Y12 fluorescence labeling was observed on GSA-B4-positive cells with low intensity. P2Y4 labeling appeared preferentially on process-bearing microglial cells (not shown). P2X2-IR (Fig. 2c,d), P2X4 (Fig. 2e,f), and P2Y6-IR (Fig. 2l,m) were found on both resting and especially on activated microglial cells, the latter being identified by the lack of processes. No immunofluorescence was observed for P2X3,5,6 receptor subtypes on either type of microglial cells before and after stab wounding.
Fig. 2Confocal images of examples of double immunofluorescence labeling to characterize the colocalization of P2X1,2,4,7 and P2Y1,6,12 receptor subtypes on a–f OX42- or g–o GSA-B4-labeled microglial cells in the NAc of rats 4 days after stab-wound injury (thin arrow process-bearing microglial cell; thick arrow activated microglial cell) (scale bars: a, b = 20 μm; c, d = 100 μm; e–i = 20 μm; j, k = 100 μm; l, m = 20 μm; n, o = 50 μm)
Injury-induced prominent expression of the P2X7 receptor was observed on microglial cells by double labeling with GSA-B4 (Fig. 2g–i) and also on astrocytes, as described earlier [29]. The two different P2X7 receptor antibodies, one directed against the extracellular domain of the rat P2X7 receptor [25] and the other against its C terminus (C-terminal P2X7) (Alomone Labs) labeled the same cells (Fig. 3a,b; thin arrow) but also different cells (Fig. 3c,d; thick arrow). Additionally, the ecto-P2X7 antibody labeled GSA-positive cells (data not shown).
Fig. 3Apoptosis or proliferation after P2X7 receptor stimulation after stab-wound injury in the NAc of rats 4 days after injury. a–d Colocalization of P2X7-IR detected by antibodies directed against the C-terminus (C-term) (b, d) and the ectodomain (a–d) of the rat P2X7. e–j Confocal images of double-labeling studies: Coexpression of active caspase 3 on single GSA-B4-positive cells (e, f) and active caspase 3 and the ecto-P2X7 receptor subtype (g, h). No double labeling of pAkT on GSA-B4-labeled microglial cells 4 days after injury (i, j) (scale bars: b–g = 20 μm). k Quantification of the number of active caspase 3-positive cells in region 1 (see Fig. 1a). Values are expressed as percentage of controls and represent the mean ± SEM of six animals per group. (*p < 0.05, vs. ACSF group; +p < 0.05,agonist vs. antagonist/agonist group; #p < 0.05, antagonist vs. antagonist/agonist group)
Possible role of the P2X7 receptor after injury in vivo
To find out whether the P2X7 receptor is related to apoptotic and/or proliferative processes after injury, we used the early apoptotic marker active caspase 3 and the proliferation marker BrdU. After BzATP microinjection, a significant increase in the number of active caspase-3-positive cells was found in comparison with ACSF-treated animals (Fig. 3k). This effect was reduced by pretreatment with the P2X7 receptor antagonist BBG. Further, BBG given alone decreased the number of active caspase 3-immunopositive cells in comparison with ACSF-treated rats (Fig. 3k).
Double immunofluorescence-labeling showed colocalization of active caspase 3 at single GSA-B4-positive cells in the NAc of rats (Fig. 3e,f). Furthermore, colocalization of the ecto-P2X7 receptor subtype on caspase-3-positive cells was also documented (Fig. 3g,h). Additionally, a possible coexpression of the anti-apoptotic and possibly proliferation-promoting pAkt on GSA-B4-positive cells was studied 4 days after injury. However, no colocalization of pAkt with GSA-B4-positive cells (Fig. 3i,j) was found by immunofluorescence labeling. Eventually, no colocalization of P2X7 and pAkt was observed to this time point (data not shown).
A possible influence of P2X7 receptor stimulation on the increase in cell number of activated and ramified microglial cells after BzATP stimulation was also studied. Changes in the number of activated microglia are shown in Fig. 1c. By quantification of ramified (resting) microglial cells in region 1 (Fig. 1a), an increase to 123.8 ± 5.3% in comparison with ACSF-treated rats was found, similar to that after treatment with 2MeSATP (124.0 ± 7.8%). For comparison, no characteristic changes were found after microinfusion of ADPβS (108.3 ± 11,6%) and α,βmeATP (101.7 ± 6.9%). Besides the effects of BzATP on the number of active and resting microglial cells, no influence of BzATP treatment on the number of GFAP (specific marker for fibrous astrocytes)-positive cells (BzATP 1.7 ± 7.3%; BBG 0.4 ± 3.0%; BzATP/BBG 0.96 ± 4.8%) was found in comparison with ACSF-treated rats. There was also no effect on the number of GFAP/BrdU double-labeled cells (proliferating cells) (BzATP 14.8 ± 4.5%; BBG 8.8 ± 11.2%; BzATP/BBG 15.9 ± 5.9%) in comparison with ACSF-treated rats.
Discussion
The results presented here demonstrate the involvement of various P2 receptors in microglial response to the placement of cannulas and microinjection, both resulting in tissue damage and thereby in high extracellular ATP concentrations [30]. Microglial cells stimulated by ATP may participate both in neurodegeneration (apoptosis and/or necrosis) and neuroprotection.
Cultured microglial cells from mouse or rat brain were shown to respond to ATP with the activation of a cationic conductance, accompanied by an increase in cytosolic Ca2+ [8], followed by changes of microglial morphology [31, 32]. The exposition of cultured microglial cells to ADP or ATP further induced membrane ruffling and markedly enhanced chemokinesis, a prerequisite to perform tissue surveillance in the brain [33].
In vivo, microglial cells are homogenously distributed within the NAc characterized by rod-shaped somata with thin and highly ramified processes extended in all directions. Injury by stab wound caused microglial cell activation around the lesion track, characterized by swollen somata and thicker processes. The additional administration of P2X and P2Y receptor agonists enhanced the occurrence of activated microglial cells detected by their BSI-IR. The observed changes suggest that the stimulation of P2X1,3 receptors has only minor relevance in the process of microglia activation, whereas in agreement with the subsequent pharmacological investigations, involvement of P2X7 and P2Y1,2,4,6,12 receptors in this process is highly probable.
Whereas P2X1–7 receptor-IR was absent in the nonaffected NAc, the expression of P2X1,2,3,4,7 receptors but not of P2X5,6 receptors was detected after the stab-wound lesion. P2X2,4,6 receptors are widely expressed in central and peripheral neurones as well as other excitable cells [22, 34, 35], and the P2X7 receptor is expressed in macrophage-type cells such as microglia but also mediates purinergic responses in astrocytes and neurons [36]. Changes in microglial expression of P2X receptors (P2X1–7) during postnatal development of the rat have been described [37]. Obviously, microglial cells are involved in long-term trophic events such as cell proliferation, differentiation, movement, and death during development [37].
Upregulation of P2X1-IR at microglial cells, as found in this study, was described at activated astrocytes after stab-wound injury in the adult rat NAc [29]. Further, the P2X2 receptor was found to be situated at activated microglia in our study and previously at astroglial cells [29]. After ischemia in vivo, P2X2 and P2X4-receptor-IR in the hippocampus became up-regulated in different cellular phenotypes [38]. P2X2 was expressed on neuronal cell bodies and fibers in hippocampal pyramidal cells, whereas intensive P2X4-IR was localized at microglial cells. Confocal microscopic analysis with OX 42 [39] at organotypic cultures showed that P2X4 and P2X7 are expressed on microglia, whereas P2X1 and P2Y1,2,12, although present in the slices, do not colocalize at microglia, and the P2X6 receptor is absent. P2X4 receptors seem to be functionally relevant for microglial chemotaxis, which was suppressed by the knockdown of the P2X4 receptor in cultured microglia by ribonucleic acid (RNA) interference through the lentivirus vector system [33].
After spinal-nerve injury, P2X4 receptor expression strikingly increased at the ipsilateral site in hyperactive microglia but not in neurons or astrocytes [40]. Intraspinal administration of P2X4 receptor antisense oligodeoxynucleotide decreased induction of P2X4 receptors and suppressed tactile allodynia after nerve injury.
The major P2X receptor expressed at microglial cells is the P2X7 receptor, characterized in several reports in vitro [13, 16, 41]. This receptor subtype appeared to be strongly coexpressed with the microglial marker GSA-B4 after the mechanical lesion used in our study, suggesting an outstanding role of this receptor in injury-induced microglial responses. It is thought to be closely related to the immune functions of microglia, such as production of inflammatory cytokines, superoxide, and nitric oxide [42]. The P2X7 receptor is reported to be little susceptible to desensitization in the presence of ATP, which makes it quite different from other purinergic receptors, including the P2X4 receptor, which can be desensitized within a few minutes [23, 43]. Up-regulation of microglial P2X7 receptors has been observed in several pathological models, such as in vivo ischemia [41, 44, 45], as well as multiple sclerosis and amyotrophic lateral sclerosis of the human spinal cord [46]. The P2X7 receptors were also specifically up-regulated around β-amyloid plaques in a mouse model of Alzheimer’s disease (Tg2576) [47] and in human proliferative vitreoretinopathy on Müller glial cells [48].
Within the P2Y receptor family, in our study, the P2Y1 receptor was markedly expressed on microglial cells of untreated rats and was up-regulated in concert with P2Y2,4,6,12 receptors after injury. In the rabbit retina, microglial cells express functional P2Y1 receptors [49]. Activation of these receptors stimulates phenotypic alterations characteristically for microglial activation.
Our data also demonstrate a strong colocalization of the P2Y6 receptor with GSA-IR. In addition to the microglial function to build a barrier between injured and healthy tissue, these cells are able to clear dead cells or dangerous debris from brain tissue. This phagocytotic process was shown to be triggered by activation of P2Y6 receptors by the endogenous agonist UDP [50].
Several authors report on ATP-induced migration of microglial cells to the affected sites of the brain [9, 10, 51–53]. Expression of the P2Y12 receptor on microglial cells after injury seems to be strongly related to the motility of this cell type. In primary cultured microglial cells, exposition to ADP or ATP induced membrane ruffling and markedly enhanced chemokinesis, which could be blocked by the P2Y12 receptor antagonist cangrelor (AR-C69931MX), suggesting that Gi/o-coupled P2Y receptors are involved [10]. The high dynamic of intact microglial processes as well as their chemotactic response to injury or local injection of ATP was shown in mouse cortex using time-lapse, two-photon laser imaging [9]. Investigations in P2Y12 receptor-deficient mice gave further support that P2Y12 receptors are required for the nucleotide-evoked chemotaxis [33, 54]. Eventually, expression of P2Y12 receptors was observed in native and axotomized facial nuclei, and the number of P2Y12-expressing cells increased following facial nerve axotomy, considered to be a crucial component in the regeneration cascades of motor neurons [55].
The published data confirm that not all known P2 receptors can be detected by their immunoreactivity. However, the mRNA of the P2X1–7 [29] and P2Y1,2,4,6,12 subtypes [27] was detectable in the NAc of untreated rats. The P2 receptors present on microglia may be differently involved in the processes of their activation, as shown by the inhibition of BSI-B4-IR using PPADS and BBG. It can be suggested by pharmacological studies that P2Y1 and P2X7 receptors may play a predominant role in the response of microglia to the consequences of a stab wound in the NAc.
Retinal detachment and application of ADPßS onto control retinas induced phenotype alterations of microglial cells (decrease of soma size, retraction of cell processes) and had no influence on microglial cell density [49]. In the stab-wound model, stimulation of P2 receptors with agonists preferring P2Y1 and P2X7 receptor subtypes promoted the morphological changes of resting to activated microglial cells.
P2X7 receptors are ATP-gated ion channels; their sustained activation leads to cytolysis in an apoptotic fashion in the microglia [56]. P2X7 receptor-mediated apoptosis involves activation of the proteolytic pathway of caspase activation, which leads to nuclear DNA damage [57]. In our study, immunocytochemistry showed expression of P2X7 and active caspase 3 at GSA-positive cells after injury without agonist application. The active caspase 3 has been defined as the main executioner of programmed cell death and as an early apoptotic marker [58]. The microinfusion of BzATP increased the number of caspase-3-positive cells, suggesting an involvement of P2X7 receptors in apoptotic changes after traumatic events. The BzATP-induced increase in ramified and activated microglia (and proliferating astrocytes) was only marginal.
The phosphoinositide-3-kinase/serine-threonine-kinase Akt (PI3-K/Akt) pathway is associated with cell proliferation, promoting cell survival and inhibiting apoptosis [59]. Because in the present study performed at 4 days after injury no coexpression of pAkt with GSA and P2X7 was found, the evidence for a preferential apoptotic pathway triggered by P2X7 receptors is unlikely.
Up-regulation of P2 receptors after mechanical injury might be both the cause and the result of the induction of apoptotic/necrotic cell death activating microglial cells with harmful or beneficial effects. The present results from in vivo investigations implicate that ATP via both P2X and P2Y receptor-stimulation acts as a signaling molecule in microglial cells in vivo. An up-regulation of P2X7-IR after injury suggests involvement of this receptor subtype in apoptotic rather than in proliferative events.

injury
p2 receptors
microglia
cns
trauma