Protective role for type 4 metabotropic glutamate receptors against ischemic brain damage

We examined the influence of type 4 metabotropic glutamate (mGlu4) receptors on ischemic brain damage using the permanent middle cerebral artery occlusion (MCAO) model in mice and the endothelin-1 (Et-1) model of transient focal ischemia in rats. Mice lacking mGlu4 receptors showed a 25% to 30% increase in infarct volume after MCAO as compared with wild-type littermates. In normal mice, systemic injection of the selective mGlu4 receptor enhancer, N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-caboxamide (PHCCC; 10 mg/kg, subcutaneous, administered once 30 minutes before MCAO), reduced the extent of ischemic brain damage by 35% to 45%. The drug was inactive in mGlu4 receptor knockout mice. In the Et-1 model, PHCCC administered only once 20 minutes after ischemia reduced the infarct volume to a larger extent in the caudate/putamen than in the cerebral cortex. Ischemic rats treated with PHCCC showed a faster recovery of neuronal function, as shown by electrocorticographic recording and by a battery of specific tests, which assess sensorimotor deficits. These data indicate that activation of mGlu4 receptors limit the development of brain damage after permanent or transient focal ischemia. These findings are promising because selective mGlu4 receptor enhancers are under clinical development for the treatment of Parkinson''s disease and other central nervous system disorders.     

Increased cerebral protein ISGylation after focal ischemia is neuroprotective

Addition of a small peptide called ISG15 is known as ISGylation, which is an ubiquitin (ub)-like posttranslational modification. We currently show that focal ischemia induced by transient middle cerebral artery occlusion (MCAO) in adult mice significantly induces cortical protein ISGylation between 6 and 24 hours reperfusion. With two-dimensional western blotting, 45 proteins were observed to be significantly increased in ISGylation (by 1.8- to 9.7-fold) after focal ischemia compared with sham control. Immunochemistry showed that ISGylated proteins are localized in neurons within the ipsilateral striatum and in astroglia within the peri-infarct cortex of ischemic mice. When subjected to transient MCAO, ISG15^−/− mice showed increased mortality, exacerbated infarction, and worsened neurologic recovery than did wild-type controls. In addition, mice lacking UBE1L (ub-activating enzyme E1-like protein, the first enzyme of the ISGylation cycle) also showed bigger infarcts when subjected to transient MCAO. Regional cerebral blood flow or other physiologic parameters were not significantly different in both knockouts compared with wild-type controls. These studies indicate that increased protein ISGylation might be an endogenous neuroprotective adaptation to minimize poststroke brain damage.     

Deficiency of PAR4 attenuates cerebral ischemia/reperfusion injury in mice

Stroke is the third leading cause of death in the USA. Antithrombotic therapy targeting platelet activation is one of the treatments for ischemic stroke. Here we investigate the role of one of the thrombin receptors, protease-activated receptor 4 (PAR4), in a mouse transient middle cerebral artery occlusion (MCAO) model. After a 60 min MCAO and 23 h reperfusion, leukocyte and platelet rolling and adhesion on cerebral venules, blood–brain barrier (BBB) permeability, and cerebral edema were compared in PAR4-deficient mice and wild-type mice. Cerebral infarction volume and neuronal death were also measured. PAR4−/− mice had more than an 80% reduction of infarct volume and significantly improved neurologic and motor function compared with wild-type mice after MCAO. Furthermore, deficiency of PAR4 significantly inhibits the rolling and adhesion of both platelets and leukocytes after MCAO. BBB disruption and cerebral edema were also attenuated in PAR4−/− mice compared with wild-type animals. The results of this investigation indicate that deficiency of PAR4 protects mice from cerebral ischemia/reperfusion (I/R) injury, partially through inhibition of platelet activation and attenuation of microvascular inflammation.     

Immune cell infiltration in malignant middle cerebral artery infarction: comparison with transient cerebral ischemia

We tested whether significant leukocyte infiltration occurs in a mouse model of permanent cerebral ischemia. C57BL6/J male mice underwent either permanent (3 or 24 hours) or transient (1 or 2 hours+22- to 23-hour reperfusion) middle cerebral artery occlusion (MCAO). Using flow cytometry, we observed ∼15,000 leukocytes (CD45^+high cells) in the ischemic hemisphere as early as 3 hours after permanent MCAO (pMCAO), comprising ∼40% lymphoid cells and ∼60% myeloid cells. Neutrophils were the predominant cell type entering the brain, and were increased to ∼5,000 as early as 3 hours after pMCAO. Several cell types (monocytes, macrophages, B lymphocytes, CD8⁺ T lymphocytes, and natural killer cells) were also increased at 3 hours to levels sustained for 24 hours, whereas others (CD4⁺ T cells, natural killer T cells, and dendritic cells) were unchanged at 3 hours, but were increased by 24 hours after pMCAO. Immunohistochemical analysis revealed that leukocytes typically had entered and widely dispersed throughout the parenchyma of the infarct within 3 hours. Moreover, compared with pMCAO, there were ∼50% fewer infiltrating leukocytes at 24 hours after transient MCAO (tMCAO), independent of infarct size. Microglial cell numbers were bilaterally increased in both models. These findings indicate that a profound infiltration of inflammatory cells occurs in the brain early after focal ischemia, especially without reperfusion.     

High susceptibility to cerebral ischemia in GFAP-null mice.

Astrocytes perform a variety of functions in the adult central nervous system (CNS) that contribute to the survival of neurons. Thus, it is likely that the activities of astrocytes affect the extent of brain damage after ischemic stroke. The authors tested this hypothesis by using a mouse ischemia model to compare the infarct volume produced in wild-type mice with that produced in mice lacking glial fibrillary acidic protein (GFAP), an astrocyte specific intermediate filament component. Astrocytes lacking GFAP have been shown to have defects in process formation, induction of the blood-brain barrier. and volume regulation; therefore, they might be compromised in their ability to protect the CNS after injury. The authors reported here that 48 hours after combined permanent middle cerebral artery occlusion (MCAO) and 15 minutes transient carotid artery occlusion (CAO) GFAP-null mice had a significantly (P < 0.001) larger cortical infarct volume (16.7 +/- 2.2 mm3) than their wild-type littermates (10.1 +/- 3.9 mm3). Laser-Doppler flowmetry revealed that the GFAP-null mice had a more extensive and profound decrease in cortical cerebral blood flow within 2 minutes after MCAO with CAO. These results indicated a high susceptibility to cerebral ischemia in GFAP-null mice and suggested an important role for astrocytes and GFAP in the progress of ischemic brain damage after focal cerebral ischemia with partial reperfusion.     

Blocking TLR2 in vivo protects against accumulation of inflammatory cells and neuronal injury in experimental stroke

Reduced infarct volume in TLR2-knockout mice compared with C57Bl/6 wild-type mice has recently been shown in experimental stroke and confirmed in this study. We now also show a significant decrease of CD11b-positive cell counts and decreased neuronal death in the ischemic hemispheres of TLR2-deficient mice compared with C57Bl/6wt mice 2 days after transient focal cerebral ischemia. To examine the potential benefit of intravascular TLR2 inhibition, C57Bl/6wt mice were treated intraarterially with TLR2-blocking anti-TLR2 antibody (clone T2.5) after 45 minutes of cerebral ischemia and compared with control antibody (isotype) treated wild-type mice. Whereas T2.5-treated mice had no reduction in infarct volumes at 48 hours after reperfusion, they did have decreased numbers of CD11b-positive inflammatory cells and decreased neuronal death compared with isotype-treated control mice. Comparison of the isotype antibody treatment to control (saline) treatment showed no effects on infarct volumes or neuronal survival. However, mice treated with the control isotype antibody had increased numbers of CD11b-positive inflammatory cells compared with saline-treated animals. Thus, antibody treatment itself (i.e., control isotype antibody, but potentially of any antibody) may have adverse effects and limit therapeutic benefit of anti-TLR2-antibody therapy. We conclude that TLR2 mediates leukocyte and microglial infiltration and neuronal death, which can be attenuated by TLR2 inhibition. The TLR2 inhibition in vivo improves neuronal survival and may represent a future stroke therapy.     

Regulatory T cells accumulate and proliferate in the ischemic hemisphere for up to 30 days after MCAO

Local and peripheral immune responses are activated after ischemic stroke. In our present study, we investigated the temporal distribution, location, induction, and function of regulatory T cells (Tregs) and the possible involvement of microglia, macrophages, and dendritic cells after middle cerebral artery occlusion (MCAO). C57BL/6J and Foxp3^EGFP transgenic mice were subjected to 30 minutes MCAO. On days 7, 14, and 30 after MCAO, Tregs and antigen presenting cells were analyzed using fluorescence activated cell sorting multicolor staining and immunohistochemistry. A strong accumulation of Tregs was observed on days 14 and 30 in the ischemic hemisphere accompanied by the elevated presence and activation of microglia. Dendritic cells and macrophages were found on each analyzed day. About 60% of Foxp3⁺ Tregs in ischemic hemispheres were positive for the proliferation marker Ki-67 on days 7 and 14 after MCAO. The transfer of naive CD4⁺ cells depleted of Foxp3⁺ Tregs into RAG1^−/− mice 1 day before MCAO did not lead to a de novo generation of Tregs 14 days after surgery. After depletion of CD25⁺ Tregs, no changes regarding neurologic outcome were detected. The sustained presence of Tregs in the brain after MCAO indicates a long-lasting immunological alteration and involvement of brain cells in immunoregulatory mechanisms.     

Spatiotemporal characteristics of postischemic hyperperfusion with respect to changes in T1, T2, diffusion,angiography, and blood–brain barrier permeability

The spatiotemporal dynamics of postischemic hyperperfusion (HP) remains incompletely understood. Diffusion, perfusion, T2, T1, angiographic, dynamic susceptibility-contrast magnetic resonance imaging (MRI) and magnetic resonance angiography were acquired longitudinally at multiple time points up to 7 days after stroke in rats subjected to 30-, 60-, and 90-minutes middle cerebral artery occlusion (MCAO). The spatiotemporal dynamics of postischemic HP was analyzed and compared with T1, T2 and blood–brain barrier (BBB) changes. No early HP within 3 hours after recanalization was observed. Late (⩾12 hours) HP was present in all animals of the 30-minute MCAO group (N=20), half of the animals in the 60-minute MCAO group (N=8), and absent in the 90-minute MCAO group (N=9). Dynamic susceptibility-contrast MRI and magnetic resonance angiography corroborated HP. Hyperperfusion preceded T2 increase in some animals, but HP and T2 changes temporally coincided in others. T2 peaked first at 24 hours whereas HP peaked at 48 hours after occlusion, and HP resolved by day 7 in most animals at which point the arteries became tortuous. Pixel-by-pixel tracking analysis showed that tissue did not infarct (migrated from core or mismatch at 30 minutes to normal at 48 hours) showed normal cerebral blood flow (CBF), whereas infarct tissue (migrated from core or mismatch at 30 minutes to infarct at 48 hours) showed exaggerated CBF, indicating that HP was associated with poor outcome.     

Glibenclamide enhances neurogenesis and improves long-term functional recovery after transient focal cerebral ischemia

Glibenclamide is neuroprotective against cerebral ischemia in rats. We studied whether glibenclamide enhances long-term brain repair and improves behavioral recovery after stroke. Adult male Wistar rats were subjected to transient middle cerebral artery occlusion (MCAO) for 90 minutes. A low dose of glibenclamide (total 0.6 μg) was administered intravenously 6, 12, and 24 hours after reperfusion. We assessed behavioral outcome during a 30-day follow-up and animals were perfused for histological evaluation. In vitro specific binding of glibenclamide to microglia increased after pro-inflammatory stimuli. In vivo glibenclamide was associated with increased migration of doublecortin-positive cells in the striatum toward the ischemic lesion 72 hours after MCAO, and reactive microglia expressed sulfonylurea receptor 1 (SUR1) and Kir6.2 in the medial striatum. One month after MCAO, glibenclamide was also associated with increased number of NeuN-positive and 5-bromo-2-deoxyuridine-positive neurons in the cortex and hippocampus, and enhanced angiogenesis in the hippocampus. Consequently, glibenclamide-treated MCAO rats showed improved performance in the limb-placing test on postoperative days 22 to 29, and in the cylinder and water-maze test on postoperative day 29. Therefore, acute blockade of SUR1 by glibenclamide enhanced long-term brain repair in MCAO rats, which was associated with improved behavioral outcome.     

Non-invasive diagnostic biomarkers for estimating the onset time of permanent cerebral ischemia

The treatments for ischemic stroke can only be administered in a narrow time-window. However, the ischemia onset time is unknown in ~30% of stroke patients (wake-up strokes). The objective of this study was to determine whether MR spectra of ischemic brains might allow the precise estimation of cerebral ischemia onset time. We modeled ischemic stroke in male ICR-CD1 mice using a permanent middle cerebral artery filament occlusion model with laser Doppler control of the regional cerebral blood flow. Mice were then subjected to repeated MRS measurements of ipsilateral striatum at 14.1 T. A striking initial increase in γ-aminobutyric acid (GABA) and no increase in glutamine were observed. A steady decline was observed for taurine (Tau), N-acetyl-aspartate (NAA) and similarly for the sum of NAA+Tau+glutamate that mimicked an exponential function. The estimation of the time of onset of permanent ischemia within 6 hours in a blinded experiment with mice showed an accuracy of 33±10 minutes. A plot of GABA, Tau, and neuronal marker concentrations against the ratio of acetate/NAA allowed precise separation of mice whose ischemia onset lay within arbitrarily chosen time-windows. We conclude that ¹H-MRS has the potential to detect the clinically relevant time of onset of ischemic stroke.     

NLRP3 deficiency ameliorates neurovascular damage in experimental ischemic stroke

Although the innate immune response to induce postischemic inflammation is considered as an essential step in the progression of cerebral ischemia injury, the role of innate immunity mediator NLRP3 in the pathogenesis of ischemic stroke is unknown. In this study, focal ischemia was induced by middle cerebral artery occlusion in NLRP3^−/−, NOX2^−/−, or wild-type (WT) mice. By magnetic resonance imaging (MRI), Evans blue permeability, and electron microscopic analyses, we found that NLRP3 deficiency ameliorated cerebral injury in mice after ischemic stroke by reducing infarcts and blood–brain barrier (BBB) damage. We further showed that the contribution of NLRP3 to neurovascular damage was associated with an autocrine/paracrine pattern of NLRP3-mediated interleukin-1β (IL-1β) release as evidenced by increased brain microvessel endothelial cell permeability and microglia-mediated neurotoxicity. Finally, we found that NOX2 deficiency improved outcomes after ischemic stroke by mediating NLRP3 signaling. This study for the first time shows the contribution of NLRP3 to neurovascular damage and provides direct evidence that NLRP3 as an important target molecule links NOX2-mediated oxidative stress to neurovascular damage in ischemic stroke. Pharmacological targeting of NLRP3-mediated inflammatory response at multiple levels may help design a new approach to develop therapeutic strategies for prevention of deterioration of cerebral function and for the treatment of stroke.     

Rivaroxaban does not increase hemorrhage after thrombolysis in experimental ischemic stroke

The management of acute ischemic stroke during anticoagulation with a novel oral anticoagulant (NOAC) is challenging because intravenous thrombolysis is contraindicated because of a putative increased risk of intracerebral hemorrhagic complications. We examined the risk of secondary postischemic hemorrhage after thrombolysis in rodents pretreated with rivaroxaban or warfarin. Mice were pretreated with either rivaroxaban (30 mg/kg), warfarin (target international normalized ratio 2 to 3) or vehicle. After 2 or 3 hours, middle cerebral artery occlusion (MCAO), mice received 9 mg/kg recombinant tissue plasminogen activator. Twenty-four hours after MCAO, secondary hemorrhage was quantified using a macroscopic hemorrhage score and hemoglobin spectrophotometry. Blood–brain barrier (BBB) permeability was measured by Evans Blue spectrofluorometry. To increase the validity of our findings, experiments were also performed using a thromboembolic model in anticoagulated rats. Infarct size did not differ among groups. Pretreatment with warfarin led to significantly more secondary hemorrhage compared with rivaroxaban and nonanticoagulated controls after 2- and 3-hour ischemia in mice as well as in rats. Blood–brain barrier permeability was significantly higher in the warfarin group compared with rivaroxaban and control. Thus, rivaroxaban in contrast to warfarin does not increase secondary hemorrhage after thrombolysis in experimental cerebral ischemia. Less effects of rivaroxaban on postischemic BBB permeability may account for this difference.     

Alteplase treatment does not increase brain injury after mechanical middle cerebral artery occlusion in the rat

Recanalization of an occluded vessel with recombinant tissue plasminogen activator is an effective strategy for treating acute ischemic stroke. Recombinant tissue plasminogen activator is administered as alteplase, a formulation containing many excipients including L-arginine, the substrate for nitric oxide production. Most studies fail to compare the effects of alteplase on brain injury to its L-arginine carrier solution. This study aimed to verify the previously reported detrimental effects of alteplase after cerebral ischemia and delineate the contribution of L-arginine. Male Wistar rats, subjected to 90 minutes of intraluminal middle cerebral artery occlusion (MCAO), were administered alteplase, the carrier solution or saline upon reperfusion. Neither alteplase nor the carrier affected cerebral blood flow (CBF) restoration throughout the first 60 minutes of reperfusion. Alteplase treatment was associated with increased mortality after MCAO. Twenty-four hours after MCAO, neurologic function and infarct volume did not differ between rats treated with alteplase, the carrier solution, or saline. Irrespective of treatment group, infarct volume was correlated with CBF during reperfusion, neuroscore, and peri-infarct depolarizations. These results suggest that alteplase treatment, independent of thrombolysis, does not cause increased ischemic injury compared with its appropriate carrier solution, supporting the continued use of alteplase in eligible ischemic stroke patients.     

Glutamate transporter type 3 knockout reduces brain tolerance to focal brain ischemia in mice

Excitatory amino-acid transporters (EAATs) transport glutamate into cells under physiologic conditions. Excitatory amino-acid transporter type 3 (EAAT3) is the major neuronal EAAT and also uptakes cysteine, the rate-limiting substrate for synthesis of glutathione. Thus, we hypothesize that EAAT3 contributes to providing brain ischemic tolerance. Male 8-week-old EAAT3 knockout mice on CD-1 mouse gene background and wild-type CD-1 mice were subjected to right middle cerebral artery occlusion for 90 minutes. Their brain infarct volumes, neurologic functions, and brain levels of glutathione, nitrotyrosine, and 4-hydroxy-2-nonenal (HNE) were evaluated. The EAAT3 knockout mice had bigger brain infarct volumes and worse neurologic deficit scores and motor coordination functions than did wild-type mice, no matter whether these neurologic outcome parameters were evaluated at 24 hours or at 4 weeks after brain ischemia. The EAAT3 knockout mice contained higher levels of HNE in the ischemic penumbral cortex and in the nonischemic cerebral cortex than did wild-type mice. Glutathione levels in the ischemic and nonischemic cortices of EAAT3 knockout mice tended to be lower than those of wild-type mice. Our results suggest that EAAT3 is important in limiting ischemic brain injury after focal brain ischemia. This effect may involve attenuating brain oxidative stress.     

Visualization of cell death in mice with focal cerebral ischemia using fluorescent annexin A5, propidium iodide,and TUNEL staining

To monitor stroke-induced brain damage and assess neuroprotective therapies, specific imaging of cell death after cerebral ischemia in a noninvasive manner is highly desirable. Annexin A5 has been suggested as a marker for imaging cell death under various disease conditions including stroke. In this study, C57BL6/N mice received middle cerebral artery occlusion (MCAO) and were injected intravenously with either active or inactive Cy5.5-annexin A5 48 hours after reperfusion. Some mice also received propidium iodide (PI), a cell integrity marker. Only in mice receiving active Cy5.5-annexin A5 were fluorescence intensities significantly higher over the hemisphere ipsilateral to MCAO than on the contralateral side. This was detected noninvasively and ex vivo 4 and 8 hours after injection. The majority of cells positive for fluorescent annexin A5 were also positive for PI and fragmented DNA as detected by terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling (TUNEL) staining. This study demonstrates the high specificity of annexin A5 for visualization of cell death in a mouse model of stroke. To our knowledge, this is the first study to compare the distribution of injected active and inactive annexin A5, PI, and TUNEL staining. It provides important information on the experimental and potential clinical applications of annexin A5-based imaging agents in stroke.     

Scavenger receptor class-A has a central role in cerebral ischemia–reperfusion injury

The innate immune response is involved in the pathophysiology of cerebral ischemia–reperfusion (I/R) injury. Recent evidence suggests that scavenger receptors have a role in the induction of innate immunity. In this study, we examined the role of scavenger receptor A (SR-A) in focal cerebral I/R injury. Both SR-A^−/− mice (n=10) and age-matched wild-type (WT) mice (n=9) were subjected to focal cerebral ischemia (60 minutes), followed by reperfusion (for 24 hours). Infarct size was determined by TTC (triphenyltetrazolium chloride) staining. The morphology of neurons in the brain sections was examined by Nissl''s staining. Activation of intracellular signaling was analyzed by western blot. Cerebral infarct size in SR-A^−/− mice was significantly reduced by 63.9% compared with WT mice after cerebral I/R. In SR-A^−/− mice, there was less neuronal damage in the hippocampus compared with WT mice. Levels of FasL, Fas, FADD, caspase-3 activity, and terminal deoynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling-positive apoptotic cells were significantly increased in WT mice after cerebral I/R, but not in SR-A^−/− mice. Cerebral I/R increased nuclear factor-κB activation in WT mice, but not in SR-A^−/− mice. These data suggest that SR-A has a central role in cerebral I/R injury and that suppression of SR-A may be a useful approach for ameliorating brain injury in stroke patients.     

Impact of genetic and renovascular chronic arterial hypertension on the acute spatiotemporal evolution of the ischemic penumbra: a sequential study with MRI in the rat

Although chronic arterial hypertension (CAH) increases the risk of stroke and the severity of the resultant lesion, it is rarely integrated in preclinical studies. Here, we analyzed the impact of CAH on the acute spatiotemporal evolution of the ischemic penumbra as defined by the perfusion-weighted imaging/diffusion-weighted imaging mismatch. Sequential 7T-MRI examinations were performed from 30 minutes up to 4 hours after permanent cerebral ischemia in genetically hypertensive rats (spontaneously hypertensive rats, SHR), renovascular-hypertensive rats (RH-WKY), and their normotensive controls (Wistar-Kyoto rats, WKY). The apparent diffusion coefficient (ADC)-defined lesion was larger in hypertensive rats than in normotensive animals as early as 30 minutes after the ischemia. The ischemic penumbra was smaller in both genetically and renovascular-hypertensive rats (at 30 minutes; SHR=66±25 mm³, RH-WKY=55±17 mm³ versus WKY=117±14 mm³; P<0.008) and there was no significant difference between the perfusion deficit and ADC lesion (mismatch definition of penumbra) as early as 90 minutes after the occlusion. Genetic hypertension and induced renovascular hypertension resulted in larger lesion and smaller penumbra that vanished rapidly. These data support the need to integrate CAH in preclinical studies relative to the treatment of stroke, as failure to do so may lead to preclinical results nonpredictive of clinical trials, which include hypertensive patients.     

Physiological variables in association with spreading depolarizations in the late phase of ischemic stroke

Physiological effects of spreading depolarizations (SD) are only well studied in the first hours after experimental stroke. In patients with malignant hemispheric stroke (MHS), monitoring of SDs is restricted to the postoperative ICU stay, typically day 2-7 post-ictus. Therefore, we investigated the role of physiological variables (temperature, intracranial pressure, mean arterial pressure and cerebral perfusion pressure) in relationship to SD during the late phase after MHS in humans. Additionally, an experimental stroke model was used to investigate hemodynamic consequences of SD during this time window. In 60 patients with MHS, the occurrence of 1692 SDs was preceded by a decrease in mean arterial pressure (−1.04 mmHg; p = .02) and cerebral perfusion pressure (−1.04 mmHg; p = .03). Twenty-four hours after middle cerebral artery occlusion in 50 C57Bl6/J mice, hypothermia led to prolonged SD-induced hyperperfusion (+2.8 min; p < .05) whereas hypertension mitigated initial hypoperfusion (−1.4 min and +18.5%Δ rCBF; p < .01). MRI revealed that SDs elicited 24 hours after experimental stroke were associated with lesion progression (15.9 vs. 14.8 mm³; p < .01). These findings of small but significant effects of physiological variables on SDs in the late phase after ischemia support the hypothesis that the impact of SDs may be modified by adjusting physiological variables.     

Single-cell resolution mapping of neuronal damage in acute focal cerebral ischemia using thallium autometallography

Neuronal damage shortly after onset or after brief episodes of cerebral ischemia has remained difficult to assess with clinical and preclinical imaging techniques as well as with microscopical methods. We here show, in rodent models of middle cerebral artery occlusion (MCAO), that neuronal damage in acute focal cerebral ischemia can be mapped with single-cell resolution using thallium autometallography (TlAMG), a histochemical technique for the detection of the K⁺-probe thallium (Tl⁺) in the brain. We intravenously injected rats and mice with thallium diethyldithiocarbamate (TlDDC), a lipophilic chelate complex that releases Tl⁺ after crossing the blood–brain barrier. We found, within the territories of the affected arteries, areas of markedly reduced neuronal Tl⁺ uptake in all animals at all time points studied ranging from 15 minutes to 24 hours after MCAO. In large lesions at early time points, areas with neuronal and astrocytic Tl⁺ uptake below thresholds of detection were surrounded by putative penumbral zones with preserved but diminished Tl⁺ uptake. At 24 hours, the areas of reduced Tl⁺uptake matched with areas delineated by established markers of neuronal damage. The results suggest the use of ²⁰¹TlDDC for preclinical and clinical single-photon emission computed tomography (SPECT) imaging of hyperacute alterations in brain K⁺ metabolism and prediction of tissue viability in cerebral ischemia.     

Thrombotic distal middle cerebral artery occlusion produced by topical FeCl3 application: a novel model suitable for intravital microscopy and thrombolysis studies

Intravital or multiphoton microscopy and laser-speckle imaging have become popular because they allow live monitoring of several processes during cerebral ischemia. Available rodent models have limitations for these experiments; e.g., filament occlusion of the proximal middle cerebral artery (MCA) is difficult to perform under a microscope, whereas distal occlusion methods may damage the MCA and the peri-arterial cortex. We found that placement of a 10% FeCl₃-soaked filter paper strip (0.3 × 1 mm²) on the duramater over the trunk of the distal MCA through a cranial window for 3 minutes induced intraarterial thrombus without damaging the peri-arterial cortex in the mouse. This caused a rapid regional cerebral blood flow decrease within 10 minutes and total occlusion of the MCA segment under the filter paper in 17±2 minutes, which resulted in a typical cortical infarct of 27±4 mm³ at 24 hours and moderate sensorimotor deficits. There was no significant hemispheric swelling or hemorrhage or mortality at 24 hours. Reperfusion was obtained in half of the mice with tissue plasminogen activator, which allowed live monitoring of clot lysis along with restoration of tissue perfusion and MCA flow. In conclusion, this relatively simple and noninvasive stroke model is easy to perform under a microscope, making it suitable for live imaging and thrombolysis studies.