Felix Schlachetzki

Normalization of Striatal Tyrosine Hydroxylase and Reversal of Motor Impairment in Experimental Parkinsonism with Intravenous Nonviral Gene Therapy and a Brain-Specific Promoter

The goal of this work was to normalize striatal tyrosine hydroxylase (TH) activity with intravenous nonviral TH gene therapy and at the same time eliminate ectopic TH gene expression in peripheral organs such as liver in the rat. TH-expression plasmids, containing either the SV40 promoter or the glial fibrillary acidic protein (GFAP) gene promoter, were globally delivered to the brain across the blood-brain barrier (BBB) after intravenous administration of pegylated immunoliposomes (PILs). The GFAP-TH- or SV40-TH-expression plasmids were encapsulated in the interior of 85-nm PILs, which were targeted across both the BBB and the neuronal cell membrane with a monoclonal antibody (mAb) to the transferrin receptor (TfR). Striatal TH activity was 98% depleted with the unilateral intracerebral injection of 6-hydroxydopamine. TH in the striatum ipsilateral to the lesion was normalized 3 days after the intravenous injection of 10 microg per rat of either the SV40-TH or the GFAP-TH plasmid DNA. Whereas the SV40-TH gene caused a 10-fold increase in hepatic TH activity, there was no increase in liver TH with the GFAP-TH gene. The GFAP-TH gene therapy caused an 82% reduction in apomorphine-induced rotation in the lesioned rats. Confocal microscopy using antibodies to TH, GFAP, and neuronal nuclei (NeuN) showed the GFAP-TH gene was selectively expressed in nigra-striatal neurons, with no expression in either cortical neurons, or astrocytes. These studies demonstrate that global delivery of exogenous genes to the brain is possible with intravenous nonviral gene transfer, and that ectopic gene expression is eliminated with the use of brain-specific gene promoters.

Amyloid-β Contributes to Blood–Brain Barrier Leakage in Transgenic Human Amyloid Precursor Protein Mice and in Humans With Cerebral Amyloid Angiopathy

Background and Purpose— Cerebral amyloid angiopathy (CAA) is a degenerative disorder characterized by amyloid-&bgr; (A&bgr;) deposition in the blood–brain barrier (BBB). CAA contributes to injuries of the neurovasculature including lobar hemorrhages, cortical microbleeds, ischemia, and superficial hemosiderosis. We postulate that CAA pathology is partially due to A&bgr; compromising the BBB. Methods— We characterized 19 patients with acute stroke with “probable CAA” for neurovascular pathology based on MRI and clinical findings. Also, we studied the effect of A&bgr; on the expression of tight junction proteins and matrix metalloproteases (MMPs) in isolated rat brain microvessels. Results— Two of 19 patients with CAA had asymptomatic BBB leakage and posterior reversible encephalopathic syndrome indicating increased BBB permeability. In addition to white matter changes, diffusion abnormality suggesting lacunar ischemia was found in 4 of 19 patients with CAA; superficial hemosiderosis was observed in 7 of 9 patients. A&bgr;40 decreased expression of the tight junction proteins claudin-1 and claudin-5 and increased expression of MMP-2 and MMP-9. Analysis of brain microvessels from transgenic mice overexpressing human amyloid precursor protein revealed the same expression pattern for tight junction and MMP proteins. Consistent with reduced tight junction and increased MMP expression and activity, permeability was increased in brain microvessels from human amyloid precursor protein mice compared with microvessels from wild-type controls. Conclusions— Our findings indicate that A&bgr; contributes to changes in brain microvessel tight junction and MMP expression, which compromises BBB integrity. We conclude that A&bgr; causes BBB leakage and that assessing BBB permeability could potentially help characterize CAA progression and be a surrogate marker for treatment response.

Cerebral ischemia-reperfusion injury in rats--a 3 T MRI study on biphasic blood-brain barrier opening and the dynamics of edema formation.

Serial magnetic resonance imaging (MRI) was performed to investigate the temporal and spatial relationship between the biphasic nature of blood–brain barrier (BBB) opening and, in parallel, edema formation after ischemia–reperfusion (I/R) injury in rats. T2-weighted imaging combined with T2-relaxometry, mainly for edema assessment, was performed at 1 h after ischemia, after reperfusion, and at 4, 24 and 48 h after reperfusion. T1-weighted imaging was performed before and after gadolinium contrast at the last three time points to assess BBB integrity. The biphasic course of BBB opening with a significant reduction in BBB permeability at 24 h after reperfusion, associated with a progressive expansion of leaky BBB volume, was accompanied by a peak ipsilateral edema formation. In addition, at 4 h after reperfusion, edema formation could also be detected at the contralateral striatum as determined by the elevated T2-values that persisted to varying degrees, indicative of widespread effects of I/R injury. The observations of this study may indicate a dynamic temporal shift in the mechanisms responsible for biphasic BBB permeability changes, with complex relations to edema formation. Stroke therapy aimed at vasogenic edema and drug delivery for neuroprotection may also be guided according to the functional status of the BBB, and these findings have to be confirmed in human stroke.

Gene therapy of the brain The trans-vascular approach

Many chronic neurologic diseases do not respond to small molecule therapeutics, and have no effective long-term therapy. Gene therapy offers the promise of an effective cure for both genetic and acquired brain disease. However, the limiting problem in brain gene therapy is delivery to brain followed by regulation of the expression of the transgene. Present day gene vectors do not cross the blood-brain barrier (BBB). Consequently, brain gene therapy requires craniotomy and the local injection of a viral gene vector. However, there are few brain disorders that can be effectively treated with local injection. Most applications of gene therapy require global expression in the brain of the exogenous gene, and this can only be achieved with a noninvasive delivery through the BBB—the trans-vascular route to brain. An additional consideration is the potential toxicity of all viral and nonviral approaches, which may either integrate into the host genome and cause insertional mutagenesis or cause inflammation in the brain. Nonviral, noninvasive gene therapy of the brain is now possible with the development of a new approach to targeting therapeutic genes to the brain following an IV administration. This approach utilizes genetically engineered molecular Trojan horses, which ferry the gene across the BBB and into neurons. Global and reversible expression of therapeutic genes in the human brain without surgery and without viral vectors is now possible.

Alert for increased long-term follow-up after carotid artery stenting: results of a prospective, randomized, single-center trial of carotid artery stenting vs carotid endarterectomy.

BACKGROUND Carotid endarterectomy (CEA) has been shown to be effective in stroke prevention for patients with symptomatic or asymptomatic carotid artery stenosis. Although several prospective randomized trials indicate that carotid artery stenting (CAS) is an alternative but not superior treatment modality, there is still a significant lack of long-term data comparing CAS with CEA. This study presents long-term results of a prospective, randomized, single-center trial. METHODS Between August 1999 and April 2002, 87 patients with a symptomatic high-grade internal carotid artery stenosis (>70%) were randomized to CAS or CEA. After a median observation time of 66 +/- 14.2 months (CAS) and 64 +/- 12.1 months (CEA), 42 patients in each group were re-evaluated retrospectively by clinical examination and documentation of neurologic events. Duplex ultrasound imaging was performed in 61 patients (32 CAS, 29 CEA), and patients with restenosis >70% were re-evaluated by angiography. RESULTS During the observation period, 23 patients (25.2%) died (10 CAS, 13 CEA), and three were lost to follow up. The incidence of strokes was higher after CAS, with four strokes in 42 CAS patients vs none in 42 CEA patients. One transient ischemic attack occurred in each group. A significantly higher rate of restenosis >70% (6 of 32 vs 0 of 29) occurred after CAS compared with CEA. Five of 32 CAS patients (15.6%) presented with high-grade (>70%) restenosis as an indication for secondary intervention or surgical stent removal, and three presented with neurologic symptoms. No CEA patients required reintervention (P < .05 vs CAS). A medium-grade (<70%) restenosis was detected in eight of 32 CAS patients (25%) and in one of 29 CEA patients (3.4%). In five of 32 CAS (15.6%) and three of 29 CEA patients (10.3%), a high-grade stenosis of the contralateral carotid artery was observed and treated during the observation period. CONCLUSION The long-term results of this prospective, randomized, single-center study revealed a high incidence of relevant restenosis and neurologic symptoms after CAS. CEA seems to be superior to CAS concerning the development of restenosis and significant prevention of stroke. However, the long-term results of the ongoing multicenter trials have to be awaited for a final conclusion.

Anemia as a risk factor for cerebral venous thrombosis? An old hypothesis revisited. Results of a prospective study.

BackgroundSeveral case reports have linked iron deficiency anemia with the occurrence of cerebral venous thrombosis (CVT) or stroke, yet, it is unclear whether this is a chance association.MethodsIn a case-control design data of whole blood count and screening for thrombophilic coagulation abnormalities of 121 prospectively identified patients with CVT and 120 healthy controls were compared. Anemia was defined as a hemoglobin (Hb) concentration of <120 g/l in females, and <130 g/l in males, severe anemia as a Hb <90 g/l. Adjusted odds ratios (OR) were calculated based on a logistic regression model treating variables with a level of significance of p ≤0.2 on univariate analysis as potential confounders.ResultsThrombophilia (OR 1.22, 95% CI 1.07-1.76, p < 0.01), severe anemia (OR 1.10, 95% CI 1.01-2.22, p < 0.05), and hypercholesterinemia (OR 1.21, 95% CI 1.04-2.57, p < 0.05) were the only independent variables associated with CVT on multivariate analysis.ConclusionSevere anemia is significantly and independently associated with CVT.

Observation on the integrity of the blood-brain barrier after microbubble destruction by diagnostic transcranial color-coded sonography.

To investigate alteration of the blood‐brain barrier from ultrasonic contrast agent destruction by diagnostic transcranial color‐coded sonography using gadolinium‐enhanced magnetic resonance imaging.

Transcranial Ultrasound from Diagnosis to Early Stroke Treatment – Part 2: Prehospital Neurosonography in Patients with Acute Stroke – The Regensburg Stroke Mobile Project

Background and Purpose: The primary aim of this study was to investigate the diagnostic accuracy and time frames for neurological and transcranial color-coded sonography (TCCS) assessments in a prehospital ‘911’ emergency stroke situation by using portable duplex ultrasound devices to visualize the bilateral middle cerebral arteries (MCAs). Methods: This study was conducted between May 2010 and January 2011. Patients who had sustained strokes in the city of Regensburg and the surrounding area in Bavaria, Germany, were enrolled in the study. After a ‘911 stroke code’ call had been dispatched, stroke neurologists with expertise in ultrasonography rendezvoused with the paramedic team at the site of the emergency. After a brief neurological assessment had been completed, the patients underwent TCCS with optional administration of an ultrasound contrast agent in cases of insufficient temporal bone windows or if the agent had acute therapeutic relevance. The ultrasound studies were performed at the site of the emergency or in the ambulance during patient transport to the admitting hospital. Relevant timelines, such as the time from the stroke alarm to patient arrival at the hospital and the duration of the TCCS, were documented, and positive and negative predictive values for the diagnosis of major MCA occlusion were assessed. Results: A total of 113 patients were enrolled in the study. MCA occlusion was diagnosed in 10 patients. In 9 of these 10 patients, MCA occlusion could be visualized using contrast-enhanced or non-contrast-enhanced TCCS during patient transport and was later confirmed using computed tomography or magnetic resonance angiography. One MCA occlusion was missed by TCCS and 1 atypical hemorrhage was misdiagnosed. Overall, the sensitivity of a ‘field diagnosis’ of MCA occlusion was 90% [95% confidence interval (CI) 55.5–99.75%] and the specificity was 98% (95% CI 92.89–99.97%). The positive predictive value was 90% (95% CI 55.5–99.75%) and the negative predictive value was 98% (95% CI 92.89–99.97%). The mean time (standard deviation) from ambulance dispatch to arrival at the patient was 12.3 min (7.09); the mean time for the TCCS examination was 5.6 min (2.2); and the overall mean transport time to the hospital was 53 min (18). Conclusion: Prehospital diagnosis of MCA occlusion in stroke patients is feasible using portable duplex ultrasonography with or without administration of a microbubble contrast agent. Prehospital neurological as well as transcranial vascular assessments during patient transport can be performed by a trained neurologist with high sensitivity and specificity, perhaps opening an additional therapeutic window for sonothrombolysis or neuroprotective strategies.

Fischer-344 rats are unsuitable for the MCAO filament model due to their cerebrovascular anatomy

Middle cerebral artery occlusion (MCAO) in Fischer-344 rats results in a small variance of infarct size. However, complications are frequent especially in aged Fisher-344 rats undergoing endovascular suture occlusion of the middle cerebral artery. Analyzing our experiences with 165 Wistar, 13 Sprague-Dawley and 10 F-344 rats, we compared the incidence of impossible thread advancement and subarachnoid hemorrhage, respectively. Magnetic resonance angiography (MRA) was applied to study the course of the internal carotid artery (ICA) in Fischer and Wistar rats. Finally, we performed a structured review of the literature from 1991 to 2005 evaluating reports on Fischer rats subjected to intraluminal filament MCAO. Complications like fruitless filament advancement or subarachnoid hemorrhage were found to be significantly more frequent in Fischer rats than in other strains. MRA revealed significantly more pronounced kinking of the ICA in F-344 than in Wistar rats. In seven publications available on filament MCAO in F-344 rats, complication rates of 50-100% were reported, corroborating our data. Surgical difficulties accompanied by high complication rates due to their cerebrovascular anatomy make Fischer rats unsuitable for filament MCAO. If the use of Fischer rats for studies on focal cerebral ischemia is indicated, other ischemia models than intraluminal suture occlusion should be chosen.

Transcranial Ultrasound from Diagnosis to Early Stroke Treatment

Background: To test whether portable duplex ultrasound devices can be used in a prehospital ‘911’ emergency situation to assess intracranial arteries. Methods: Non-contrast-enhanced transcranial duplex ultrasound studies were performed either immediately at the site of the emergency (i.e. private home) or after transfer into the emergency helicopter/ambulance vehicle. Results: A total of 25 patients were enrolled. In 5/25 cases, intracranial vessels could not be visualized due to insufficient quality of the temporal bone window. In 20/25 cases, bilateral visualization and Doppler flow measurements of the middle cerebral artery could be assessed in a mean time less than 2 min. Conclusion: Emergency assessment of intracranial arteries using portable duplex ultrasound devices is feasible shortly after arrival at the patient’s site.