Michael S. Dittmar

External Carotid Artery Territory Ischemia Impairs Outcome in the Endovascular Filament Model of Middle Cerebral Artery Occlusion in Rats

BACKGROUND AND PURPOSE Middle cerebral artery occlusion (MCAO) by an intraluminal filament is a widely accepted animal model of focal cerebral ischemia. In this procedure, cutting of the external carotid artery (ECA) is a prerequisite for thread insertion. However, the implications of ECA transsection have not yet been described. METHODS After 90 minutes of filament MCAO or sham surgery, rats were evaluated for up to 14 days in terms of body weight development, core temperature, and motor performance. Repeated in vivo MRI of the head and neck was performed for quantification of brain edema and infarct volume. The temporal muscles were histologically analyzed postmortem. RESULTS In 47% of all rats, ischemic tissue damage to the ipsilateral ECA area, including temporal, lingual, and pharyngeal musculature, was detectable by MRI. Histology of temporal muscles confirmed acute ischemic myopathy. Animals with ECA territory ischemia (ECA-I) showed delayed body weight development and poorer recovery of motor function. There was no difference in the extent of brain edema or final cerebral lesion size between ECA-I-affected and unaffected rats. CONCLUSIONS Filament MCAO was complicated by the consequences of ECA ischemia in approximately half of all rats. Impaired mastication and swallowing functions restricted ingestion and resulted in postsurgical body weight loss and worse motor performance. Impaired cerebral microperfusion resulting from dehydration and reduced spontaneous motor activity resulting from reduced food and water uptake might have contributed to poorer neurological recovery in ECA ischemic rats. Thus, adverse effects caused by extracerebral ischemia with potential impact on outcome have to be considered in this stroke model.

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.

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.

Thrombolysis for Stroke in the Elderly

Introduction. Systemic thrombolysis with intravenous recombinant tissue plasminogen activator (rtPA) for acute ischemic stroke had been licensed for patients up to 75 years in age in many European countries and was recently extended to 80 years. This age restriction results from the potential higher risk of cerebral bleeding in the elderly. The major rtPA trials included only 42 patients above 80 years showing a potential benefit from treatment. Further data is still rare.Methods. Using our stroke database we identified all patients beyond 75 years with middle cerebral artery ischemia treated with intravenous rtPA in our stroke unit from 02/1999 until 07/2004. Clinical course and outcome until day 5 in addition to mortality after 3 and 6 months were analysed.Results. Twenty-nine patients (80.8 ± 4 years, 16 of them over 80 years old) met the inclusion criteria representing 21.2% of those receiving thrombolytic therapy. The median NIH-SS score on admission was 14 points. On day 5 after thrombolysis, 13/29 showed a good recovery (NIH-SS improvement ≥4 pts). The remaining exhibited only small or no benefit (n = 11), deterioration (n = 3) or died (n = 2). A total of 3/29 patients developed non-symptomatic parenchymal hemorrhage or hemorrhagic transformation. One patient died due to space-occupying cerebral hemorrhage. Extracerebral bleeding was found in 3/29 requiring substitution in one. One other died for primary cardiac reasons. Median NIH-SS on day 5 was 10 points. Mortality after 3 and 6 months was 20.7%. We did not find factors predicting clinical outcome. Most importantly, there was no significant difference regarding outcome in patients 76–80 vs. 81–87 years old.Discussion. Intravenous rtPA resulted in good neurological in-hospital outcome in almost 45% and six-months survival of almost 80% of the patients beyond 75 years. In 10.3% non-symptomatic and in 3.4% symptomatic cerebral bleeding was found. Thus, seniors beyond 75 and even beyond 80 years in good medical condition may benefit from systemic treatment with rtPA. Prospective studies are needed to clarify which part of the senior population might be most eligible for systemic thrombolysis.

Neurovascular protection by targeting early blood–brain barrier disruption with neurotrophic factors after ischemia–reperfusion in rats

The ‘new penumbra’ concept imbues the transition between injury and repair at the neurovascular unit with profound implications for selecting the appropriate type and timing of neuroprotective interventions. In this conceptual study, we investigated the protective effects of pigment epithelium-derived factor (PEDF) and compared them with the properties of epidermal growth factor (EGF) in a rat model of ischemia–reperfusion injury. We initiated a delayed intervention 3 hours after reperfusion using equimolar amounts of PEDF and EGF. These agents were then administered intravenously for 4 hours following reperfusion after 1 hour of focal ischemia. Magnetic resonance imaging indices were characterized, and imaging was performed at multiple time points post reperfusion. PEDF and EGF reduced lesion volumes at all time points as observed on T2-weighted images (T2-LVs). In addition PEDF selectively attenuated lesion volume expansion at 48 hours after reperfusion and persistently modulated blood–brain barrier (BBB) permeability at all time points. Intervention with peptides is suspected to cause edema formation at distant regions. The observed T2-LV reduction and BBB modulation by these trophic factors is probably mediated through a number of diverse mechanisms. A thorough evaluation of neurotrophins is still necessary to determine their time-dependent contributions against injury and their modulatory effects on repair after stroke.

Flow‐cytometric measurement of respiratory burst in rat polymorphonuclear granulocytes: Comparison of four cell preparation procedures, and concentration–response evaluation of soluble stimulants

Polymorphonuclear neutrophils (PMNs) contribute to organ injury in sepsis, stroke, and other diseases. Evaluation of the oxidative burst by flow cytometry (FCM) is frequently applied to examine PMN status in humans, but rarely in rats. We established a method to assess granulocyte activation in rats by means of FCM analysis of oxidative burst. Two methods for PMN isolation involving Histopaque separation were investigated, and additionally two whole blood techniques. In addition, the concentration–response relation of the stimulants fMLP, PMA, TNF‐α, and LPS has been determined, both as sole stimulants and for priming. A novel technique with diluted rat whole blood proved to be most appropriate for PMN preparation. One micromolar PMA and fMLP, respectively, are effective concentrations for PMN stimulation in rat whole blood. Priming with 0.1 μg/ml TNF‐α and 1 μg/ml LPS, respectively, resulted in optimal additional stimulation. This study defined the appropriate conditions for evaluating the reactive oxygen derivate production in rat PMNs by flow cytometry. The rapid, simple, and reliable cell preparation procedure of whole blood dilution that preserves cell integrity and requires only small sample quantities. This is the first systematic dose–response evaluation of soluble stimulants of neutrophil respiratory burst in rats.

Adverse Effects of the Intraluminal Filament Model of Middle Cerebral Artery Occlusion

To the Editor: Animal models of ischemic stroke are of major importance for experimental stroke research. Comprehensive knowledge of the methodological aspects of the different stroke models available is crucial for data interpretation and correlation to human stroke. We therefore appreciate the recent work of Gerriets et al on complications in different models of focal ischemia in rats, taking advantage of high-resolution magnetic resonance imaging (MRI) and magnetic resonance angiography.1 In addition to their findings of subarachnoid hemorrhage and hypothalamic infarction as a cause of hyperthermia, the appearance of ipsilateral masticatory hyperintensities in early MRI associated with temporal muscle necrosis (Figure) has also been identified recently as a complication of the intraluminal filament model of middle cerebral artery occlusion (MCAO). These lesions resulted in impaired body weight evolution and delayed restoration of neurological function in Wistar rats.2 This is neither a laboratory-specific nor a rat strain-specific problem, as can be learned from …

The role of ECA transection in the development of masticatory lesions in the MCAO filament model.

In the intraluminal suture model of middle cerebral artery occlusion (MCAO) in the rat, lesions of the masticator muscles associated with impaired functional outcome occur. We evaluated the role of external carotid artery (ECA) transection. We assessed whether isolated interruption of an arterial or a venous connection to the ECA territory was sufficient to induce masticatory hypoperfusion and lesions. We also evaluated a direct access to the common carotid artery (CCA) with subsequent vascular closure with regard to its feasibility, frequency of masticatory lesions, complications, and cerebral ischemia. Cerebral and masticatory lesions and perfusion deficits were assessed by in vivo magnetic resonance imaging (MRI). Vessel patency was evaluated using computerized tomography angiography and histology. An interruption of arterial blood flow led to masticatory hypoperfusion. Masticatory lesions occurred in 6% of the rats. Access to and closure of the CCA were feasible in all animals, leading to moderate or severe vessel stenosis in 20%, and intraarterial thrombosis in 25% of the rats. Reproducible cerebral infarctions were obtained in all animals. In 24% of the rats, hyperintense MRI signal changes were observed in the ipsilateral temporal muscle. Thus, the induction of masticatory hypoperfusion and lesions by arterial transection supports the role of the ECA in this context. Direct access to the CCA with subsequent vessel closure led to stenosis in most animals. Preservation of ECA continuity was not suitable to fully prevent masticatory lesions.

Evaluation of a novel algorithm for primary mass casualty triage by paramedics in a physician manned EMS system: a dummy based trial

BackgroundThe Amberg-Schwandorf Algorithm for Primary Triage (ASAV) is a novel primary triage concept specifically for physician manned emergency medical services (EMS) systems. In this study, we determined the diagnostic reliability and the time requirements of ASAV triage.MethodsSeven hundred eighty triage runs performed by 76 trained EMS providers of varying professional qualification were included into the study. Patients were simulated using human dummies with written vital signs sheets. Triage results were compared to a standard solution, which was developed in a modified Delphi procedure. Test performance parameters (e.g. sensitivity, specificity, likelihood ratios (LR), under-triage, and over-triage) were calculated. Time measurements comprised the complete triage and tagging process and included the time span for walking to the subsequent patient. Results were compared to those published for mSTaRT. Additionally, a subgroup analysis was performed for employment status (career/volunteer), team qualification, and previous triage training.ResultsFor red patients, ASAV sensitivity was 87%, specificity 91%, positive LR 9.7, negative LR 0.139, over-triage 6%, and under-triage 10%. There were no significant differences related to mSTaRT. Per patient, ASAV triage required a mean of 35.4 sec (75th percentile 46 sec, 90th percentile 58 sec). Volunteers needed slightly more time to perform triage than EMS professionals. Previous mSTaRT training of the provider reduced under-triage significantly. There were significant differences in time requirements for triage depending on the expected triage category.ConclusionsThe ASAV is a specific concept for primary triage in physician governed EMS systems. It may detect red patients reliably. The test performance criteria are comparable to that of mSTaRT, whereas ASAV triage might be accomplished slightly faster. From the data, there was no evidence for a clinically significant reliability difference between typical staffing of mobile intensive care units, patient transport ambulances, or disaster response volunteers. Up to now, there is no clinical validation of either triage concept. Therefore, reality based evaluation studies are needed.German AbstractHintergrundDer Amberg-Schwandorf-Algorithmus für die Vorsichtung (ASAV) ist ein neues Ablaufschema für die Vorsichtung durch Nicht-Ärzte beim Massenanfall von Verletzten und speziell für notarztbasierte Rettungsdienstsysteme entwickelt. In der vorliegenden Arbeit untersuchten wir die Testgütekriterien und den Zeitbedarf für die Vorsichtung mittels ASAV.Methodenfür die Studie wurden 780 Sichtungsvorgänge von 76 Probanden unterschiedlicher Qualifikation ausgewertet. Als Patienten wurden Dummies, ausgestattet mit einer schriftlichen Zustandsbeschreibung verwendet. Die Sichtungsergebnisse wurden mit einer Musterlösung verglichen, welche in einem modifizierten Delphi-Prozess erstellt wurden. Daraus wurden Testgütekriterien wie Sensitivität, Spezifität, Wahrscheinlichkeitsverhältnisse (LR), Über- und Untertriage berechnet. Die Zeitmessung umfasste den kompletten Sichtungs- und Sichtungskennzeichnungsvorgang einschließlich der Wegezeit zum nächsten Patienten. Die Ergebnisse wurden zu den für mSTaRT veröffentlichten Daten verglichen. Zusätzlich wurde eine vordefinierte Subgruppenanalyse in Bezug auf Beschäftigungsverhältnis (Haupt- vs. Ehrenamt), Teamzusammensetzung und vorherige Sichtungsausbildung durchgeführt.ErgebnisseBezogen auf die roten Patienten betrug die Sensitivität 87%, die Spezifität 91%, die positive LR 9,7, die negative LR 0,139, die Übertriage 6% und die Untertriage 10%. Signifikante Unterschiede zu mSTaRT konnten nicht nachgewiesen werden. Die Sichtung mittels ASAV benötigte durchschnittlich 35,4 Sek. pro Patient (75. Perzentil 46 Sek., 90. Perzentil 58 Sek.). Ehrenamtliche benötigten geringfügig länger für die Vorsichtung. Eine vorausgehende Ausbildung in einem anderen Sichtungskonzept reduzierte die Untertriage signifikant. Je nach erwarteter Sichtungskategorie unterschied sich der Zeitbedarf für die Vorsichtung signifikant.SchlussfolgerungenDer ASAV ist ein Konzept für die Vorsichtung speziell für notarztbasierte Rettungsdienstsysteme. Durch ASAV scheinen sich die roten Patienten verlässlich identifizieren zu lassen. Die Testgütekriterien sind vergleichbar mit den für mSTaRT publizierten Werten, wobei ASAV etwas weniger Zeit in Anspruch zu nehmen scheint. Unsere Daten ergaben keinen signifikanten Unterschied bei der Vorsichtungs-Reliabilität zwischen typischen Besatzungen von Rettungstransportwägen, Krankentransportwägen oder ehrenamtlichen Katastrophenschutzeinheiten. Gegenwärtig ist kein Vorsichtungsalgorithmus klinisch validiert. Daher sind weitere Untersuchungen an Realeinsätzen notwendig.

Application of Clinical Scanners in Rats: Experimental Carotid Imaging Using Magnetic Resonance Imaging, Spiral Computed Tomography, and Color Duplex Ultrasound

Background and Purpose. Noninvasive small animal imaging allows for reduction of the required numbers of animals in research by providing the possibility of long‐term follow‐up at various time points. Additionally, correlation to the investigated respective human disease is possible as equivalent equipment is employed. The authors therefore evaluate feasibility and potential of color duplex sonography, computed tomography angiography (CTA), and magnetic resonance angiography (MRA) by the use of clinical scanners for carotid artery imaging in rats. Methods. Male Wistar rats (n = 17) were subjected to color duplex sonography, CTA, and MRA of the common carotid artery (CCA) and the carotid bifurcation. Clinical scanners were used for the experiments and optimal parameter settings evaluated accounting for the different size of the animals. The applied imaging methods were analyzed in regard to image quality and practicability in laboratory settings. Results. The CCA could be clearly displayed by all imaging modalities in all rats. Duplex sonography provided distinct images and reproducible basic functional information. CTA and MRA provided distinct images of the CCA and the carotid bifurcation in both axial and reconstructed 3‐dimensional images. The authors further describe different indications for these imaging methods regarding spatial resolution, acquisition times, possible scanning range, and application of contrast agent. Conclusions. Color duplex sonography, CTA, and MRAare all feasible methods for imaging of the carotid arteries in rats. Images of sufficient clarity and resolution could be obtained by the use of clinical scanners, yielding information about vessel size, direction of blood flow, and adjacent structures. Further studies need to be performed that Address investigations of pathological conditions such as flow disturbances or vessel stenosis.