Maureen Walberer

Noninvasive Quantification of Brain Edema and the Space-Occupying Effect in Rat Stroke Models Using Magnetic Resonance Imaging

Background and Purpose— Brain edema is a life-threatening consequence of stroke and leads to an extension of the affected tissue. The space-occupying effect due to brain edema can be quantified in rat stroke models with the use of MRI. The present study was performed to test 2 hypotheses: (1) Can quantification of the space-occupying effect due to brain edema serve as a noninvasive measure for brain water content? (2) Does morphometric assessment of brain swelling allow determination of true infarct size on MRI after correction for the space-occupying effect of edema? Methods— Thirty rats were subjected to permanent suture middle cerebral artery occlusion. MRI was performed after 6 or 24 hours, and hemispheric swelling was assessed morphometrically. Interobserver and intraobserver agreements were determined for MRI measurements. In study I, the space-occupying effect due to brain edema was correlated with the absolute brain water content by the wet/dry method. In study II, lesion volumes corrected and uncorrected for edema were calculated on MRI and on TTC staining and compared. Results— Interobserver and intraobserver agreements for MRI measurements were excellent (r ≥0.97). Brain water content and hemispheric swelling correlated well after 6 and 24 hours (r ≥0.95). Corrected lesion volumes correlated with r =0.78 between TTC staining and MRI. Without edema correction, lesion volumes were overestimated by 20.3% after 6 hours and by 29.6% after 24 hours of ischemia. Conclusions— Morphometric assessment of hemispheric swelling on MRI can determine the increase in absolute brain water content noninvasively and can also provide ischemic lesion volumes corrected for brain edema.

Complications and Pitfalls in Rat Stroke Models for Middle Cerebral Artery Occlusion A Comparison Between the Suture and the Macrosphere Model Using Magnetic Resonance Angiography

Background and Purpose— Investigating focal cerebral ischemia requires animal models that are relevant to human stroke. Complications and side effects are common among these models. The present study describes potential pitfalls in 3 techniques for middle cerebral artery occlusion (MCAO) in rats using magnetic resonance imaging (MRI) and magnetic resonance angiography (MRA). Methods— Rats were subjected to temporary MCAO for 90 minutes using the suture technique (group I; n=10) or to permanent MCAO using the suture technique (group II; n=10) or the macrosphere technique (group III; n=10). Clinical evaluation was performed after 3 hours and 24 hours. After 24 hours, animals underwent MRI and MRA to determine lesion size and the intracranial vascular status. Results— Hemispheric lesion volume was significantly smaller in group I (14.6%) compared with groups II (35.2%; P<0.01) and III (21.3%; P<0.05). Two animals (1 each in group II and III) did not demonstrate neurological deficits and had no lesion on MRI and a patent MCA main stem on MRA. Subarachnoid hemorrhage was detected in 2 animals (1 each in group I and II). MRA indicated a patent MCA main stem in 2 animals (group II), although both rats displayed neurological deficits. Hypothalamic infarction with subsequent pathological hyperthermia was detected in all animals in group II and in 1 rat in group III. Conclusions— Model failures occurred frequently in all groups. MRI and MRA helps to identify animals that need to be excluded from experimental stroke studies.

Neuroinflammation Extends Brain Tissue at Risk to Vital Peri-Infarct Tissue: A Double Tracer [11C]PK11195- and [18F]FDG-PET Study

Focal cerebral ischemia elicits strong inflammatory responses involving activation of resident microglia and recruitment of monocytes/macrophages. These cells express peripheral benzodiazepine receptors (PBRs) and can be visualized by positron emission tomography (PET) using [11C]PK11195 that selectively binds to PBRs. Earlier research suggests that transient ischemia in rats induces increased [11C]PK11195 binding within the infarct core. In this study, we investigated the expression of PBRs during permanent ischemia in rats. Permanent cerebral ischemia was induced by injection of macrospheres into the middle cerebral artery. Multimodal imaging 7 days after ischemia comprised (1) magnetic resonance imaging that assessed the extent of infarcts; (2) [18F]-2-fluoro-2-deoxy-d-glucose ([18F]FDG)-PET characterizing cerebral glucose transport and metabolism; and (3) [11C]PK11195-PET detecting neuroinflammation. Immunohistochemistry verified ischemic damage and neuroinflammatory processes. Contrasting with earlier data for transient ischemia, no [11C]PK11195 binding was found in the infarct core. Rather, permanent ischemia caused increased [11C]PK11195 binding in the normoperfused peri-infarct zone (mean standard uptake value (SUV): 1.93 ± 0.49), colocalizing with a 60% increase in the [18F]FDG metabolic rate constant with accumulated activated microglia and macrophages. These results suggest that after permanent focal ischemia, neuroinflammation occurring in the normoperfused peri-infarct zone goes along with increased energy demand, therefore extending the tissue at risk to areas adjacent to the infarct.

Noninvasive Imaging of Endogenous Neural Stem Cell Mobilization In Vivo Using Positron Emission Tomography

Neural stem cells reside in two major niches in the adult brain [i.e., the subventricular zone (SVZ) and the dentate gyrus of the hippocampus]. Insults to the brain such as cerebral ischemia result in a physiological mobilization of endogenous neural stem cells. Since recent studies showed that pharmacological stimulation can be used to expand the endogenous neural stem cell niche, hope has been raised to enhance the brains own regenerative capacity. For the evaluation of such novel therapeutic approaches, longitudinal and intraindividual monitoring of the endogenous neural stem cell niche would be required. However, to date no conclusive imaging technique has been established. We used positron emission tomography (PET) and the radiotracer 3′-deoxy-3′-[18F]fluoro-l-thymidine ([18F]FLT) that enables imaging and measuring of proliferation to noninvasively detect endogenous neural stem cells in the normal and diseased adult rat brain in vivo. This method indeed visualized neural stem cell niches in the living rat brain, identified as increased [18F]FLT-binding in the SVZ and the hippocampus. Focal cerebral ischemia and subsequent damage of the blood–brain barrier did not interfere with the capability of [18F]FLT-PET to visualize neural stem cell mobilization. Moreover, [18F]FLT-PET allowed for an in vivo quantification of increased neural stem cell mobilization caused by pharmacological stimulation or by focal cerebral ischemia. The data suggest that noninvasive longitudinal monitoring and quantification of endogenous neural stem cell activation in the brain is feasible and that [18F]FLT-PET could be used to monitor the effects of drugs aimed at expanding the neural stem cell niche.

Neuroprotective Effects of MK-801 in Different Rat Stroke Models for Permanent Middle Cerebral Artery Occlusion Adverse Effects of Hypothalamic Damage and Strategies for Its Avoidance

BACKGROUND AND PURPOSE Permanent middle cerebral artery occlusion (MCAO) with the use of the suture technique causes hypothalamic damage with subsequent hyperthermia, which can confound neuroprotective drug studies. In the present study the neuroprotective effects of dizocilpine (MK-801) were compared in different permanent MCAO models with and without hypothalamic damage and hyperthermia. METHODS Sixty Sprague-Dawley rats were treated with MK-801 or placebo, beginning 15 minutes before MCAO, and assigned to the following groups: suture MCAO (group I), macrosphere MCAO without hypothalamic damage (group II), or macrosphere MCAO with intentionally induced hypothalamic infarction (group III). Body temperature was measured at 3, 6, and 24 hours. Lesion size was determined after 24 hours (2,3,5-triphenyltetrazolium chloride staining). RESULTS Hypothalamic damage was present in animals in group I and was intentionally induced in group III with the use of a modified macrosphere MCAO technique. Body temperature was significantly increased 3, 6, and 24 hours after MCAO in these 2 groups of animals. Hypothalamic damage and subsequent hyperthermia could be avoided effectively by limiting the number of macrospheres (group II). MK-801 provided a highly significant neuroprotective effect in group II but not in groups I and III. CONCLUSIONS Hypothalamic damage with subsequent hyperthermia masked the neuroprotective effect of MK-801. This side effect can be avoided by using the macrosphere MCAO technique with a limited number of spheres. This model therefore may be more appropriate to study the effects of neuroprotective drugs in permanent focal cerebral ischemia than the suture method.

Multi-session transcranial direct current stimulation (tDCS) elicits inflammatory and regenerative processes in the rat brain.

Transcranial direct current stimulation (tDCS) is increasingly being used in human studies as an adjuvant tool to promote recovery of function after stroke. However, its neurobiological effects are still largely unknown. Electric fields are known to influence the migration of various cell types in vitro, but effects in vivo remain to be shown. Hypothesizing that tDCS might elicit the recruitment of cells to the cortex, we here studied the effects of tDCS in the rat brain in vivo. Adult Wistar rats (n = 16) were randomized to either anodal or cathodal stimulation for either 5 or 10 consecutive days (500 µA, 15 min). Bromodeoxyuridine (BrdU) was given systemically to label dividing cells throughout the experiment. Immunohistochemical analyses ex vivo included stainings for activated microglia and endogenous neural stem cells (NSC). Multi-session tDCS with the chosen parameters did not cause a cortical lesion. An innate immune response with early upregulation of Iba1-positive activated microglia occurred after both cathodal and anodal tDCS. The involvement of adaptive immunity as assessed by ICAM1-immunoreactivity was less pronounced. Most interestingly, only cathodal tDCS increased the number of endogenous NSC in the stimulated cortex. After 10 days of cathodal stimulation, proliferating NSC increased by ∼60%, with a significant effect of both polarity and number of tDCS sessions on the recruitment of NSC. We demonstrate a pro-inflammatory effect of both cathodal and anodal tDCS, and a polarity-specific migratory effect on endogenous NSC in vivo. Our data suggest that tDCS in human stroke patients might also elicit NSC activation and modulate neuroinflammation.

Experimental stroke: ischaemic lesion volume and oedema formation differ among rat strains (a comparison between Wistar and Sprague–Dawley rats using MRI)

Investigating focal cerebral ischaemia requires animal models that are relevant to human stroke. This study was designed to evaluate the influence of early reperfusion and choice of rat strains on infarct volume and oedema formation. Thirty-six Wistar and Sprague–Dawley rats were subjected to temporary middle cerebral artery occlusion (MCAO) for 90 min (groups I and II) or to permanent MCAO (groups III and IV) using the suture technique. Ischaemic lesion volume and oedema formation were quantified 24 h after MCAO using 7T-magnetic resonance imaging (MRI). Impact of rat strains: Reperfusion led to significant larger ischaemic lesion volumes in Wistar rats as compared to Sprague–Dawley rats (P<0.0005). Oedema formation was similar in both rat strains. Permanent MCAO led to significantly larger ischaemic lesion volumes in Sprague–Dawley rats (P<0.05). Oedema formation, however, was significantly more accentuated in Wistar rats (P<0.005). Impact of reperfusion: Reperfusion did not cause any changes in ischaemic lesion volume in Wistar rats. Oedema formation, however, was significantly reduced (P<0.0005). In Sprague–Dawley rats, reperfusion caused a significant reduction of ischaemic lesion volume (P<0.00005), but did not modify oedema formation. These findings emphasize the critical importance of rat strain differences in experimental stroke research.

Signaling mechanism of extracellular RNA in endothelial cells

Extracellular RNA has been shown to induce vascular endothelial growth factor (VEGF)‐dependent hyperpermeability in vivo as well as in vitro. Studies were performed to investigate the mechanism of these effects. For permeability studies primary cultures of porcine brain‐derived microvascular endothelial cells (BMECs) and for all other analytical studies the human brain endothelial cell line HCMEC/D3 or human umbilical vein endothelial cells (HUVECs) were used. RNA, but not DNA, initiated signaling events by binding of VEGF to neuropilin‐1, followed by VEGF‐R2 phosphorylation, activation of phospholipase C (PLC), and intracellular release of Ca2+. Activation of these pathways by RNA also resulted in the release of von Willebrand Factor from Weibel‐Palade bodies. Pretreat‐ment of cells with heparinase totally abrogated the RNA‐induced permeability changes, whereas RNA together with VEGF completely restored VEGF‐R2‐mediated hyperpermeability. Although poly:IC increased the interleukin‐6 release via activation of toll‐like receptor‐3 (TLR‐3), permeability changes mediated by poly:IC or RNA remained unchanged after blocking TLR‐3 or NF‐kB activation. These results indicate that extracellular RNA serves an important cofactor function to engage VEGF for VEGF‐R2‐dependent signal transduction, reminiscent of the coreceptor mechanism mediated by proteoglycans, which might be of relevance for the mobilization and cellular activities of RNA‐binding cytokines in general.—Fischer, S.,Nishio, M., Peters, S. C., Tschernatsch, M., Walberer, M., Weidemann, S., Heidenreich, R., Couraud, P. O., Weksler, B. B., Romero, I. A., Gerriets, T., Preissner, K. T. Signaling mechanism of extracellular RNA in endothelial cells. FASEBJ. 23, 2100–2109 (2007)

Edema formation in the hyperacute phase of ischemic stroke. Laboratory investigation.

OBJECT Brain edema formation is a serious complication of ischemic stroke and can lead to mechanical compression of adjacent brain structures, cerebral herniation, and death. Furthermore, the space-occupying effect of edema impairs regional cerebral blood flow (rCBF), which is particularly important in the penumbra phase of stroke. In the present study, the authors evaluated the natural course of edema formation in the hyperacute phase of focal cerebral ischemia. METHODS Middle cerebral artery occlusion (MCAO) or a sham procedure was performed in rats within an MR imaging unit (in-bore occlusion). Both pre- and postischemic images could be compared on a pixel-by-pixel basis. The T2 relaxation time (T2RT), a marker for brain water content, was measured in regions of interest. RESULTS A significant increase in the T2RT was detectable as early as 20-45 minutes after MCAO. At this early time point the midline shift (MLS) amounted to 0.214 +/- 0.092 cm in the MCAO group and 0.061 +/- 0.063 cm in the sham group (p < 0.007). The T2RT and MLS increased linearly thereafter. Evans blue dye was intravenously injected in additional animals 20 and 155 minutes after MCAO. Extravasation of the dye was visible in all animals, indicating increased permeability of the blood-brain barrier. CONCLUSIONS Vasogenic brain edema occurs much earlier than expected following permanent MCAO and leads to MLS and mechanical compression of adjacent brain structures. Since compression effects can impair rCBF, early edema formation can significantly contribute to infarct formation and thus represents a promising target for neuroprotection.

Aggravation of infarct formation by brain swelling in a large territorial stroke: a target for neuroprotection?

OBJECT In territorial stroke vasogenic edema formation leads to elevated intracranial pressure (ICP) and can cause herniation and death. Brain swelling further impairs collateral blood flow to the ischemic penumbra and causes mechanical damage to adjacent brain structures. In the present study the authors sought to quantify the impact of this space-occupying effect on ischemic lesion formation. METHODS Wistar rats were assigned to undergo bilateral craniectomy or a sham operation and then were subjected to temporary middle cerebral artery occlusion (MCAO) for 90 minutes. A clinical evaluation and 7-T MR imaging studies were performed 5 and 24 hours after MCAO. The absolute brain water content was determined at 24 hours by using the wet/dry method. RESULTS Bilateral craniectomy before MCAO led to a drastic reduction in lesion volume at both imaging time points (p < 0.0001). Ischemic lesion volume was 2.7- and 2.3-fold larger in sham-operated animals after 5 and 24 hours, respectively. Clinical scores were likewise better in rats that had undergone craniectomy (p < 0.05). After 24 hours the midline shift differed significantly between the 2 groups (p < 0.001), but not after 5 hours. The relation between brain water content and ischemic lesion volume as well as the T2 relaxation time within the infarcted area was not different between the groups (p > 0.05). CONCLUSIONS The data indicated that collateral damage caused by the space-occupying effect of a large MCA territory stroke contributes seriously to ischemic lesion formation. The elimination of increased ICP thus must be regarded as a highly neuroprotective measure, rather than only a life-saving procedure to prevent cerebral herniation. Further clinical trials should reveal the neuroprotective potential of surgical and pharmacological ICP-lowering therapeutic approaches.