Christiane Frahm

Identification of Ischemic Regions in a Rat Model of Stroke

Background Investigations following stroke first of all require information about the spatio-temporal dimension of the ischemic core as well as of perilesional and remote affected tissue. Here we systematically evaluated regions differently impaired by focal ischemia. Methodology/Principal Findings Wistar rats underwent a transient 30 or 120 min suture-occlusion of the middle cerebral artery (MCAO) followed by various reperfusion times (2 h, 1 d, 7 d, 30 d) or a permanent MCAO (1 d survival). Brains were characterized by TTC, thionine, and immunohistochemistry using MAP2, HSP72, and HSP27. TTC staining reliably identifies the infarct core at 1 d of reperfusion after 30 min MCAO and at all investigated times following 120 min and permanent MCAO. Nissl histology denotes the infarct core from 2 h up to 30 d after transient as well as permanent MCAO. Absent and attenuated MAP2 staining clearly identifies the infarct core and perilesional affected regions at all investigated times, respectively. HSP72 denotes perilesional areas in a limited post-ischemic time (1 d). HSP27 detects perilesional and remote impaired tissue from post-ischemic day 1 on. Furthermore a simultaneous expression of HSP72 and HSP27 in perilesional neurons was revealed. Conclusions/Significance TTC and Nissl staining can be applied to designate the infarct core. MAP2, HSP72, and HSP27 are excellent markers not only to identify perilesional and remote areas but also to discriminate affected neuronal and glial populations. Moreover markers vary in their confinement to different reperfusion times. The extent and consistency of infarcts increase with prolonged occlusion of the MCA. Therefore interindividual infarct dimension should be precisely assessed by the combined use of different markers as described in this study.

Plasticity of rat central inhibitory synapses through GABA metabolism

1 The production of the central inhibitory transmitter GABA (γ‐aminobutyric acid) varies in response to different patterns of activity. It therefore seems possible that GABA metabolism can determine inhibitory synaptic strength and that presynaptic GABA content is a regulated parameter for synaptic plasticity. 2 We altered presynaptic GABA metabolism in cultured rat hippocampal slices using pharmacological tools. Degradation of GABA by GABA‐transaminase (GABA‐T) was blocked by γ‐vinyl‐GABA (GVG) and synthesis of GABA through glutamate decarboxylase (GAD) was suppressed with 3‐mercaptopropionic acid (MPA). We measured miniature GABAergic postsynaptic currents (mIPSCs) in CA3 pyramidal cells using the whole‐cell patch clamp technique. 3 Elevated intra‐synaptic GABA levels after block of GABA‐T resulted in increased mIPSC amplitude and frequency. In addition, tonic GABAergic background noise was enhanced by GVG. Electron micrographs from inhibitory synapses identified by immunogold staining for GABA confirmed the enhanced GABA content but revealed no further morphological alterations. 4 The suppression of GABA synthesis by MPA had opposite functional consequences: mIPSC amplitude and frequency decreased and current noise was reduced compared with control. However, we were unable to demonstrate the decreased GABA content in biochemical analyses of whole slices or in electron micrographs. 5 We conclude that the transmitter content of GABAergic vesicles is variable and that postsynaptic receptors are usually not saturated, leaving room for up‐regulation of inhibitory synaptic strength. Our data reveal a new mechanism of plasticity at central inhibitory synapses and provide a rationale for the activity‐dependent regulation of GABA synthesis in mammals.

Attenuated Inflammatory Response in Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) Knock-Out Mice following Stroke

Background Triggering receptor expressed on myeloid cells-2 (TREM2) is a microglial surface receptor involved in phagocytosis. Clearance of apoptotic debris after stroke represents an important mechanism to re-attain tissue homeostasis and thereby ensure functional recovery. The role of TREM2 following stroke is currently unclear. Methods and Results As an experimental stroke model, the middle cerebral artery of mice was occluded for 30 minutes with a range of reperfusion times (duration of reperfusion: 6 h/12 h/24 h/2 d/7 d/28 d). Quantitative PCR (qPCR) revealed a greatly increased transcription of TREM2 after stroke. We subsequently analyzed the expression of pro-inflammatory cytokines, chemokines and their receptors in TREM2-knockout (TREM2-KO) mice via qPCR. Microglial activation (CD68, Iba1) and CD3-positive T-cell invasion were analyzed via qPCR and immunohistochemistry. Functional consequences of TREM2 knockout were assessed by infarct volumetry. The acute inflammatory response (12 h reperfusion) was very similar between TREM2-KO mice and their littermate controls. However, in the sub-acute phase (7 d reperfusion) following stroke, TREM2-KO mice showed a decreased transcription of pro-inflammatory cytokines TNFα, IL-1α and IL-1β, associated with a reduced microglial activity (CD68, Iba1). Furthermore, TREM2-KO mice showed a reduced transcription of chemokines CCL2 (MCP1), CCL3 (MIP1α) and the chemokine receptor CX3CR1, followed by a diminished invasion of CD3-positive T-cells. No effect on the lesion size was observed. Conclusions Although we initially expected an exaggerated pro-inflammatory response following ablation of TREM2, our data support a contradictory scenario that the sub-acute inflammatory reaction after stroke is attenuated in TREM2-KO mice. We therefore conclude that TREM2 appears to sustain a distinct inflammatory response after stroke.

LAMINAR DIFFERENCE IN GABA UPTAKE AND GAT-1 EXPRESSION IN RAT CA1

1 The axonal plexus of most hippocampal interneurons is restricted to certain strata within the target region. This lamination suggests a possible functional heterogeneity of inhibitory synapses between different interneurons and CA1 pyramidal cells. 2 We therefore compared inhibitory postsynaptic potentials (IPSPs) and currents (IPSCs) in CA1 pyramidal cells, which were evoked from two stimulation sites (stratum oriens and stratum radiatum). Stimulation in stratum oriens yielded faster decaying IPSPs and IPSCs than stimulation in stratum radiatum. 3 IPSP and IPSC kinetics were regulated by GABA uptake in both layers as indicated by the prolongation of the signals under tiagabine, a GAT‐1 (neuronal GABA plasma membrane transporter)‐specific GABA‐uptake blocker. However, the effect of tiagabine was significantly more pronounced following stimulation in stratum radiatum than in stratum oriens (prolongation of IPSC half‐decay time by 167 vs. 115 %, respectively). 4 In situ hybridization with antisense mRNA for the GABA‐synthesizing enzyme glutamate decarboxylase (GAD65/67) and the GABA transporter GAT‐1 showed that the proportion of interneurons expressing GAT‐1 was lower in stratum oriens than in stratum radiatum/lacunosum‐moleculare. 5 From these functional and molecular data we conclude that the regulation of IPSP and IPSC kinetics in CA1 pyramidal cells by neuronal GABA uptake differs between layers. Our findings suggest that this laminar difference is caused by a lower expression of GAT‐1 in interneurons in stratum oriens than in stratum radiatum/lacunosum‐moleculare.

Downregulation of Potassium Chloride Cotransporter KCC2 After Transient Focal Cerebral Ischemia

Background and Purpose— The potassium chloride cotransporter 2 (KCC2) is the main neuronal chloride extruder in the adult nervous system. Therefore, KCC2 is responsible for an inwardly directed electrochemical gradient of chloride that leads to hyperpolarizing GABA-mediated responses. Under some pathophysiological conditions, GABA has been reported to be depolarizing because of a downregulation of KCC2. This is the first study to our knowledge analyzing the expression of KCC2 after a focal cerebral ischemia. Methods— Mild and severe ischemia were induced in rats by a transient occlusion of the middle cerebral artery for 30 and 120 minutes, respectively. KCC2 mRNA and protein expression were studied in the ischemic hemisphere after different reperfusion times (2 hour, 1 day, 7 days, 30 days, 168 days) by using quantitative polymerase chain reaction, Western blotting, and immunohistological staining. Results— We found a substantial decrease of KCC2 mRNA and protein levels in the ischemic hemisphere, with a stronger downregulation of KCC2 after severe vs mild ischemia. Long-term surviving cells expressing KCC2 could be detected in the infarct core. These cells were identified as GABAergic interneurons mainly expressing parvalbumin. Conclusions— Our study revealed a substantial neuron-specific downregulation of KCC2 after focal cerebral ischemia.

Attenuated Inflammatory Response in Aged Mice Brains following Stroke

Background Increased age is a major risk factor for stroke incidence, post-ischemic mortality, and severe and long-term disability. Stroke outcome is considerably influenced by post-ischemic mechanisms. We hypothesized that the inflammatory response following an ischemic injury is altered in aged organisms. Methods and Results To that end, we analyzed the expression pattern of pro-inflammatory cytokines (TNF, IL-1α, IL-1β, IL-6), anti-inflammatory cytokines (IL-10, TGFβ1), and chemokines (Mip-1α, MCP-1, RANTES) of adult (2 months) and aged (24 months) mice brains at different reperfusion times (6 h, 12 h, 24 h, 2 d, 7 d) following transient occlusion of the middle cerebral artery. The infarct size was assessed to monitor possible consequences of an altered inflammatory response in aged mice. Our data revealed an increased neuro-inflammation with age. Above all, we found profound age-related alterations in the reaction to stroke. The response of pro-inflammatory cytokines (TNF, and IL-1β) and the level of chemokines (Mip-1α, and MCP-1) were strongly diminished in the aged post-ischemic brain tissue. IL-6 showed the strongest age-dependent decrease in its post-ischemic expression profile. Anti-inflammatory cytokines (TGFβ1, and IL-10) revealed no significant age dependency after ischemia. Aged mice brains tend to develop smaller infarcts. Conclusion The attenuated inflammatory response to stroke in aged animals may contribute to their smaller infarcts. The results presented here highlight the importance of using aged animals to investigate age-associated diseases like stroke, and should be considered as a major prerequisite in the development of age-adjusted therapeutic interventions.

GAD and GABA transporter (GAT-1) mRNA expression in the developing rat hippocampus.

Synaptic inhibition in the mammalian central nervous system is mostly mediated by GABA (gamma-aminobutyric acid). Inhibitory interneurons can be identified by staining for glutamate decarboxylase (GAD), the key enzyme which produces the transmitter. After release, GABA is removed from the extracellular space by specific transporters which are localized at the presynaptic endings of interneurons, in adjacent glial processes and, possibly, also in the postsynaptic target cell membranes. The GABAergic system undergoes profound functional and structural changes during the first 2 weeks of postnatal development, including migration of interneurons and changes in the level of expression and subcellular distribution of GABA transporters. We therefore analyzed the distribution of mRNA coding for GAD and GAT-1 (the main neuronal GABA transporter) in the developing rat hippocampus. Our data show that both transcripts are present in putative interneurons from the first postnatal day and exhibit a largely similar distribution throughout postnatal ontogenesis, with some specific differences in certain hippocampal subfields. Quantification of stained somata confirmed the postnatal redistribution of putative interneurons in the area dentata from dendritic layers towards the hilus. We also found a general staining of principal cell layers for both probes, which differs with postnatal age and between GAD and GAT-1 mRNA. Together, our data reveal a profound reorganization of the GABAergic system in the rat hippocampus during the first weeks of postnatal development.

Efficacy of background GABA uptake in rat hippocampal slices

GABA uptake is crucial for the termination of inhibitory synaptic events. In addition, GABA transporters may also control the level of diffusely distributed GABA in the extracellular space. We analysed this function by superfusing rat hippocampal slices with different concentrations of GABA. Whole-cell patch clamp recordings of CA1 pyramidal cells revealed small increases in chloride conductance at 5–10 μM GABA which increased dramatically upon addition of the GABA uptake blocker tiagabine. Tiagabine alone induced a significant chloride conductance indicating that spontaneous release of GABA in hippocampal slices is neutralized by GAT-1, the main hippocampal GABA transporter. Thus, GAT-1 clears the extracellular space in the hippocampus from diffusely distributed GABA with high efficacy.

Presence of γ-aminobutyric acid transporter mRNA in interneurons and principal cells of rat hippocampus

After release, neurotransmitters are removed from the extracellular space by high-affinity uptake. Specific sodium-dependent transporters serve this function for the inhibitory transmitter gamma-aminobutyric acid (GABA). However, it is largely unknown to which proportion GABA is taken up by GABAergic interneurons, glia cells or principal neurons. We analyzed the distribution of mRNA for the main GABA-transporter subtype in the hippocampus, GAT-1, in adult rats. Most interneurons were strongly stained for GAT-1 mRNA, indicating re-uptake by the GABA-releasing cells. Surprisingly, prominent signals for GAT-1 were also found throughout the principal cell layers (granule and pyramidal cells). These data indicate that GABA transporters may be present in non-GABAergic projection cells of the rat hippocampus which contribute to the clearance of GABA from the extracellular space.

Branched-chain amino acid catabolism is a conserved regulator of physiological ageing

Ageing has been defined as a global decline in physiological function depending on both environmental and genetic factors. Here we identify gene transcripts that are similarly regulated during physiological ageing in nematodes, zebrafish and mice. We observe the strongest extension of lifespan when impairing expression of the branched-chain amino acid transferase-1 (bcat-1) gene in C. elegans, which leads to excessive levels of branched-chain amino acids (BCAAs). We further show that BCAAs reduce a LET-363/mTOR-dependent neuro-endocrine signal, which we identify as DAF-7/TGFβ, and that impacts lifespan depending on its related receptors, DAF-1 and DAF-4, as well as ultimately on DAF-16/FoxO and HSF-1 in a cell-non-autonomous manner. The transcription factor HLH-15 controls and epistatically synergizes with BCAT-1 to modulate physiological ageing. Lastly and consistent with previous findings in rodents, nutritional supplementation of BCAAs extends nematodal lifespan. Taken together, BCAAs act as periphery-derived metabokines that induce a central neuro-endocrine response, culminating in extended healthspan.