Severity Assessment in Rats Undergoing Subarachnoid Hemorrhage Induction by Endovascular Perforation or Corresponding Sham Surgery.

INTRODUCTION: Animal models for preclinical research of subarachnoid hemorrhage (SAH) are widely used as much of the pathophysiology remains unknown. However, the burden of these models inflicted on the animals is not well characterized. The European directive requires severity assessment-based allocation to categories. Up to now, the classification into predefined categories is rather subjective and often without underlying scientific knowledge. We therefore aimed at assessing the burden of rats after SAH or the corresponding sham surgery to provide a scientific assessment. METHODS: We performed a multimodal approach, using different behavior tests, clinical and neurological scoring, and biochemical markers using the common model for SAH of intracranial endovascular filament perforation in male Wistar rats. Up to 7 days after surgery, animals with SAH were compared to sham surgery and to a group receiving only anesthesia and analgesia. RESULTS: Sham surgery (n = 15) and SAH (n = 16) animals showed an increase in the clinical score the first days after surgery, indicating clinical deterioration, while animals receiving only anesthesia without surgery (n = 5) remained unaffected. Body weight loss occurred in all groups but was more pronounced and statistically significant only after surgery. The analysis of burrowing, open field (total distance, erections), balance beam, and neuroscore showed primarily an effect of the surgery itself in sham surgery and SAH animals. Only concerning balance beam and neuroscore, a difference was visible between sham surgery and SAH. The outcome of the analysis of systemic and local inflammatory parameters and of corticosterone in blood and its metabolites in feces was only robust in animals suffering from larger bleedings. Application of principal component analysis resulted in a clear separation of sham surgery and SAH animals from their respective baseline as well as from the anesthesia-only group at days 1 and 3, with the difference between sham surgery and SAH being not significant. DISCUSSION/CONCLUSION: To our knowledge, we are the first to publish detailed clinical score sheet data combined with advanced behavioral assessment in the endovascular perforation model for SAH in rats. The tests chosen here clearly depict an impairment of the animals within the first days after surgery and are consequently well suited for assessment of the animals' suffering in the model. A definitive classification into one of the severity categories named by the EU directive is yet pending and has to be performed in the future by including the assessment data from different neurological and nonneurological disease models.

Failed Neuroprotection of Combined Inhibition of L-Type and ASIC1a Calcium Channels with Nimodipine and Amiloride.

Effective pharmacological neuroprotection is one of the most desired aims in modern medicine. We postulated that a combination of two clinically used drugs-nimodipine (L-Type voltage-gated calcium channel blocker) and amiloride (acid-sensing ion channel inhibitor)-might act synergistically in an experimental model of ischaemia, targeting the intracellular rise in calcium as a pathway in neuronal cell death. We used organotypic hippocampal slices of mice pups and a well-established regimen of oxygen-glucose deprivation (OGD) to assess a possible neuroprotective effect. Neither nimodipine (at 10 or 20 µM) alone or in combination with amiloride (at 100 µM) showed any amelioration. Dissolved at 2.0 Vol.% dimethyl-sulfoxide (DMSO), the combination of both components even increased cell damage (p = 0.0001), an effect not observed with amiloride alone. We conclude that neither amiloride nor nimodipine do offer neuroprotection in an in vitro ischaemia model. On a technical note, the use of DMSO should be carefully evaluated in neuroprotective experiments, since it possibly alters cell damage.

Pericytes are involved in the pathogenesis of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy.

OBJECTIVE: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common inherited small-vessel disease, is associated with vascular aggregation of mutant Notch3 protein, dysfunction of cerebral vessels, and dementia. Pericytes, perivascular cells involved in microvascular function, express Notch3. Therefore, we hypothesize that these cells may play a role in the pathogenesis of CADASIL. METHODS: Two-, 7-, and 12-month-old CADASIL mutant mice (TgNotch3(R169C) ) and wild-type controls were examined regarding Notch3 aggregation in pericytes, the coverage of cerebral vessels by pericytes, pericyte numbers, capillary density, blood-brain barrier (BBB) integrity, astrocytic end-feet, and the expression of astrocytic gap junction and endothelial adherens junction protein using immunostaining and Western blot analysis. In addition, we examined cerebrovascular CO2 reactivity using laser Doppler fluxmetry and in vivo microscopy. RESULTS: With increasing age, mutated Notch3 aggregated around pericytes and smooth muscle cells. Notch3 aggregation caused significant reduction of pericyte number and coverage of capillaries by pericyte processes (p < 0.01). These changes were associated with detachment of astrocytic end-feet from cerebral microvessels, leakage of plasma proteins, reduction in expression of endothelial adherens junction protein, and reduced microvascular reactivity to CO2 . Smooth muscle cells were not affected by Notch3 accumulation. INTERPRETATION: Our results show that pericytes are the first cells affected by Notch3 aggregation in CADASIL mice. Pericyte pathology causes opening of the BBB and microvascular dysfunction. Therefore, protecting pericytes may represent a novel therapeutic strategy for vascular dementia.

Secondary Ischemia Assessment in Murine and Rat Preclinical Subarachnoid Hemorrhage Models: A Systematic Review.

BACKGROUND: Delayed cerebral ischemia represents a significant contributor to death and disability following aneurysmal subarachnoid hemorrhage. Although preclinical models have shown promising results, clinical trials have consistently failed to replicate the success of therapeutic strategies. The lack of standardized experimental setups and outcome assessments, particularly regarding secondary vasospastic/ischemic events, may be partly responsible for the translational failure. The study aims to delineate the procedural characteristics and assessment modalities of secondary vasospastic and ischemic events, serving as surrogates for clinically relevant delayed cerebral ischemia, in recent rat and murine subarachnoid hemorrhage models. METHODS AND RESULTS: We conducted a systematic review of rat and murine in vivo subarachnoid hemorrhage studies (published: 2016-2020) using delayed cerebral ischemia/vasospasm as outcome parameters. Our analysis included 102 eligible studies. In murine studies (n=30), the endovascular perforation model was predominantly used, while rat studies primarily employed intracisternal blood injection to mimic subarachnoid hemorrhage. Particularly, the injection models exhibited considerable variation in injection volume, rate, and cerebrospinal fluid withdrawal. Peri-interventional monitoring was generally inadequately reported across all models, with body temperature and blood pressure being the most frequently documented parameters (62% and 34%, respectively). Vasospastic events were mainly assessed through microscopy of large cerebral arteries. In 90% of the rat and 86% of the murine studies, only male animals were used. CONCLUSIONS: Our study underscores the substantial heterogeneity in procedural characteristics and outcome assessments of experimental subarachnoid hemorrhage research. To address these challenges, drafting guidelines for standardization and ensuring rigorous control of methodological and experimental quality by funders and journals are essential. REGISTRATION: URL: https://www.crd.york.ac.uk/prospero/; Unique identifier: CRD42022337279.

Essential role of interleukin-6 in post-stroke angiogenesis.

Ambivalent effects of interleukin-6 on the pathogenesis of ischaemic stroke have been reported. However, to date, the long-term actions of interleukin-6 after stroke have not been investigated. Here, we subjected interleukin-6 knockout (IL-6(-/-)) and wild-type control mice to mild brain ischaemia by 30-min filamentous middle cerebral artery occlusion/reperfusion. While ischaemic tissue damage was comparable at early time points, IL-6(-/-) mice showed significantly increased chronic lesion volumes as well as worse long-term functional outcome. In particular, IL-6(-/-) mice displayed an impaired angiogenic response to brain ischaemia with reduced numbers of newly generated endothelial cells and decreased density of perfused microvessels along with lower absolute regional cerebral blood flow and reduced vessel responsivity in ischaemic striatum at 4 weeks. Similarly, the early genomic activation of angiogenesis-related gene networks was strongly reduced and the ischaemia-induced signal transducer and activator of transcription 3 activation observed in wild-type mice was almost absent in IL-6(-/-) mice. In addition, systemic neoangiogenesis was impaired in IL-6(-/-) mice. Transplantation of interleukin-6 competent bone marrow into IL-6(-/-) mice (IL-6(chi)) did not rescue interleukin-6 messenger RNA expression or the early transcriptional activation of angiogenesis after stroke. Accordingly, chronic stroke outcome in IL-6(chi) mice recapitulated the major effects of interleukin-6 deficiency on post-stroke regeneration with significantly enhanced lesion volumes and reduced vessel densities. Additional in vitro experiments yielded complementary evidence, which showed that after stroke resident brain cells serve as the major source of interleukin-6 in a self-amplifying network. Treatment of primary cortical neurons, mixed glial cultures or immortalized brain endothelia with interleukin 6-induced robust interleukin-6 messenger RNA transcription in each case, whereas oxygen-glucose deprivation did not. However, oxygen-glucose deprivation of organotypic brain slices resulted in strong upregulation of interleukin-6 messenger RNA along with increased transcription of key angiogenesis-associated genes. In conclusion, interleukin-6 produced locally by resident brain cells promotes post-stroke angiogenesis and thereby affords long-term histological and functional protection.

Pericytes in capillaries are contractile in vivo, but arterioles mediate functional hyperemia in the mouse brain.

Modern functional imaging techniques of the brain measure local hemodynamic responses evoked by neuronal activity. Capillary pericytes recently were suggested to mediate neurovascular coupling in brain slices, but their role in vivo remains unexplored. We used two-photon microscopy to study in real time pericytes and the dynamic changes of capillary diameter and blood flow in the cortex of anesthetized mice, as well as in brain slices. The thromboxane A(2) analog, 9,11-dideoxy-9α,11α-methanoepoxy Prostaglandin F2α (U46619), induced constrictions in the vicinity of pericytes in a fraction of capillaries, whereas others dilated. The changes in vessel diameter resulted in changes in capillary red blood cell (RBC) flow. In contrast, during brief epochs of seizure activity elicited by local administration of the GABA(A) receptor antagonist, bicuculline, capillary RBC flow increased without pericyte-induced capillary diameter changes. Precapillary arterioles were the smallest vessels to dilate, together with penetrating and pial arterioles. Our results provide in vivo evidence that pericytes can modulate capillary blood flow in the brain, which may be important under pathological conditions. However, our data suggest that precapillary and penetrating arterioles, rather than pericytes in capillaries, are responsible for the blood flow increase induced by neural activity.

Effect of isolated intracranial hypertension on cerebral perfusion within the phase of primary disturbances after subarachnoid hemorrhage in rats.

Introduction: Elevated intracranial pressure (ICP) and blood components are the main trigger factors starting the complex pathophysiological cascade following subarachnoid hemorrhage (SAH). It is not clear whether they independently contribute to tissue damage or whether their impact cannot be differentiated from each other. We here aimed to establish a rat intracranial hypertension model that allows distinguishing the effects of these two factors and investigating the relationship between elevated ICP and hypoperfusion very early after SAH. Methods: Blood or four different types of fluids [gelofusine, silicone oil, artificial cerebrospinal fluid (aCSF), aCSF plus xanthan (CX)] were injected into the cisterna magna in anesthetized rats, respectively. Arterial blood pressure, ICP and cerebral blood flow (CBF) were continuously measured up to 6 h after injection. Enzyme-linked immunosorbent assays were performed to measure the pro-inflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) in brain cortex and peripheral blood. Results: Silicone oil injection caused deaths of almost all animals. Compared to blood, gelofusine resulted in lower peak ICP and lower plateau phase. Artificial CSF reached a comparable ICP peak value but failed to reach the ICP plateau of blood injection. Injection of CX with comparable viscosity as blood reproduced the ICP course of the blood injection group. Compared with the CBF course after blood injection, CX induced a comparable early global ischemia within the first minutes which was followed by a prompt return to baseline level with no further hypoperfusion despite an equal ICP course. The inflammatory response within the tissue did not differ between blood or blood-substitute injection. The systemic inflammation was significantly more pronounced in the CX injection group compared with the other fluids including blood. Discussion: By cisterna magna injection of blood substitution fluids, we established a subarachnoid space occupying rat model that exactly mimicked the course of ICP in the first 6 h following blood injection. Fluids lacking blood components did not induce the typical prolonged hypoperfusion occurring after blood-injection in this very early phase. Our study strongly suggests that blood components rather than elevated ICP play an important role for early hypoperfusion events in SAH.

Opening a window to the acutely injured brain: Simultaneous retinal and cerebral vascular monitoring in rats.

Many recent research projects have described typical chronic changes in the retinal vasculature for diverse neurovascular and neurodegenerative disorders such as stroke or Alzheimer's disease. Unlike cerebral vasculature, retinal blood vessels can be assessed non-invasively by retinal vessel analysis. To date, there is only a little information about potential simultaneous reactions of retinal and cerebral vessels in acute neurovascular diseases. The field of applications of retinal assessment could significantly be widened if more information about potential correlations between those two vascular beds and the feasibility of non-invasive retinal vessel analysis in acute neurovascular disease were available. Here, we present our protocol for the simultaneous assessment of retinal and cerebral vessels in an acute setting in anesthetized rats using a non-invasive retinal vessel analyzer and a superficial tissue imaging system for laser speckle contrast analysis via a closed bone window. We describe the experimental set-up in detail, outline the pitfalls of repeated retinal vessel analyses in an experimental set-up of several hours, and address issues that arise from the simultaneous use of two different assessment tools. Finally, we demonstrate the robustness and variability of the reactivity of retinal vessels to hypercapnia at baseline as well as their reproducibility over time using two anesthetic protocols common for neurovascular research. In summary, the procedures described in this protocol allow us to directly compare retinal and cerebral vascular beds and help to substantiate the role of the retina as a "window to the brain."

Procedural and Methodological Quality in Preclinical Stroke Research-A Cohort Analysis of the Rat MCAO Model Comparing Periods Before and After the Publication of STAIR/ARRIVE.

The translation of preclinical stroke research into successful human clinical trials remains a challenging task. The first Stroke Therapy Academic Industry Roundtable (STAIR) recommendations for preclinical research and several other guidelines were published to address these challenges. Most guidelines recommend the use of physiological monitoring to detect the occurrence of undesired pathologies such as subarachnoid hemorrhage and to limit the variability of the infarct volume and-therefore-homogenize the experimental result for complete reporting particularly with respect to transparency and methodological rigor. From the years 2009 and 2019, 100 published articles each using a rat stroke model were analyzed to quantify parameters related to anesthesia, physiological monitoring, stroke model type, ischemia verification, and overall study quality over time. No significant difference in the frequency of cerebral blood flow (CBF) measurements over time (28/34% for 2009/2019) was found. Notably, significantly fewer studies reported temperature, blood pressure, and blood gas monitoring data in 2019 compared to 2009. On the other hand, an increase in general study quality parameters (e.g., randomization, reporting of approval) was seen. In conclusion, the frequency of periinterventional monitoring has decreased over time. Some general methodological quality aspects, however, partially have increased. CBF measurement-the gold standard for ischemia verification-was applied rarely. Despite the growing recognition of current guidelines such as STAIR and ARRIVE (both widely approved in 2019) reporting, methods and procedures mostly do not follow these guidelines. These deficits may contribute to the translational failure of preclinical stroke research in search for neuroprotective therapies.

Neuroprotective Effects of the Inert Gas Argon on Experimental Traumatic Brain Injury In Vivo with the Controlled Cortical Impact Model in Mice.

Argon has shown neuroprotective effects after traumatic brain injury (TBI) and cerebral ischemia in vitro and in focal cerebral ischemia in vivo. The purpose of this study is to show whether argon beneficially impacts brain contusion volume (BCV) as the primary outcome parameter, as well as secondary outcome parameters, such as brain edema, intracranial pressure (ICP), neurological outcome, and cerebral blood flow (CBF) in an in-vivo model. Subjects were randomly assigned to either argon treatment or room air. After applying controlled cortical impact (CCI) onto the dura with 8 m/s (displacement 1 mm, impact duration 150 ms), treatment was administered by a recovery chamber with 25%, 50%, or 75% argon and the rest being oxygen for 4 h after trauma. Two control groups received room air for 15 min and 24 h, respectively. Neurological testing and ICP measurements were performed 24 h after trauma, and brains were removed to measure secondary brain damage. The primary outcome parameter, BCV, and the secondary outcome parameter, brain edema, were not significantly reduced by argon treatment at any concentration. There was a highly significant decrease in ICP at 50% argon (p = 0.001), and significant neurological improvement (beamwalk missteps) at 25% and 50% argon (p = 0.01; p = 0.049 respectively) compared to control.

Burrowing behaviour of rats: Strain differences and applicability as well-being parameter after intracranial surgery.

In mice, burrowing is considered a species-typical parameter for assessing well-being, while this is less clear in rats. This exploratory study evaluated burrowing behaviour in three rat strains during training and in the direct postoperative phase after complex intracranial surgery in different neuroscience rat models established at Hannover Medical School or Aachen University Hospital. Male Crl:CD (SD; n = 18), BDIX/UlmHanZtm (BDIX; n = 8) and RjHan:WI (Wistar; n = 35) rats were individually trained to burrow gravel out of a tube on four consecutive days. Thereafter, BDIX rats were subjected to intracranial injection of BT4Ca cells and tumour resection (rat glioma model), SD rats to injection of 6-hydroxydopamine (6-OHDA) or vehicle (rat Parkinson's disease model) and Wistar rats to endovascular perforation or sham surgery (rat subarachnoid haemorrhage (SAH) model). Burrowing was retested on the day after surgery. During training, BDIX rats burrowed large amounts (mean of 2370 g on the fourth day), while SD and Wistar rats burrowed less gravel (means of 846 and 520 g, respectively). Burrowing increased significantly during training only in Wistar rats. Complex surgery, that is, tumour resection (BDIX), 6-OHDA injection (SD) and endovascular perforation or sham surgery for SAH (Wistar) significantly reduced burrowing and body weight, while simple stereotactic injection of tumour cells or vehicle did not affect burrowing. Despite the training, burrowing differed between the strains. In the direct postoperative phase, burrowing was reduced after complex surgery, indicating reduced well-being. Reduced burrowing was accompanied with postoperative weight loss, a validated and recognised quantitative measure for severity assessment.

Vascular Reactivity to Hypercapnia Is Impaired in the Cerebral and Retinal Vasculature in the Acute Phase After Experimental Subarachnoid Hemorrhage.

Objective: Impaired cerebral blood flow (CBF) regulation, such as reduced reactivity to hypercapnia, contributes to the pathophysiology after aneurysmal subarachnoid hemorrhage (SAH), but temporal dynamics in the acute phase are unknown. Featuring comparable molecular regulation mechanisms, the retinal vessels participate in chronic and subacute stroke- and SAH-associated vessel alterations in patients and can be studied non-invasively. This study is aimed to characterize the temporal course of the cerebral and retinal vascular reactivity to hypercapnia in the acute phase after experimental SAH and compare the potential degree of impairment. Methods: Subarachnoid hemorrhage was induced by injecting 0.5 ml of heparinized autologous blood into the cisterna magna of male Wistar rats using two anesthesia protocols [isoflurane/fentanyl n = 25 (Sham + SAH): Iso-Group, ketamine/xylazine n = 32 (Sham + SAH): K/X-Group]. CBF (laser speckle contrast analysis) and physiological parameters were measured continuously for 6 h. At six predefined time points, hypercapnia was induced by hypoventilation controlled via blood gas analysis, and retinal vessel diameter (RVD) was determined non-invasively. Results: Cerebral reactivity and retinal reactivity in Sham groups were stable with only a slight attenuation after 2 h in RVD of the K/X-Group. In the SAH Iso-Group, cerebral and retinal CO2 reactivity compared to baseline was immediately impaired starting at 30 min after SAH (CBF p = 0.0090, RVD p = 0.0135) and lasting up to 4 h (p = 0.0136, resp. p = 0.0263). Similarly, in the K/X-Group, cerebral CO2 reactivity was disturbed early after SAH (30 min, p = 0.003) albeit showing a recovery to baseline after 2 h while retinal CO2 reactivity was impaired over the whole observation period (360 min, p = 0.0001) in the K/X-Group. After normalization to baseline, both vascular beds showed a parallel behavior regarding the temporal course and extent of impairment. Conclusion: This study provides a detailed temporal analysis of impaired cerebral vascular CO2 reactivity starting immediately after SAH and lasting up to 6 h. Importantly, the retinal vessels participate in these acute changes underscoring the promising role of the retina as a potential non-invasive screening tool after SAH. Further studies will be required to determine the correlation with functional outcomes.

Retinal Vessel Responses to Flicker Stimulation Are Impaired in Ca v 2.3-Deficient Mice-An in-vivo Evaluation Using Retinal Vessel Analysis (RVA).

Objective: Metabolic demand increases with neuronal activity and adequate energy supply is ensured by neurovascular coupling (NVC). Impairments of NVC have been reported in the context of several diseases and may correlate with disease severity and outcome. Voltage-gated Ca2+-channels (VGCCs) are involved in the regulation of vasomotor tone. In the present study, we compared arterial and venous responses to flicker stimulation in Cav2.3-competent (Cav2.3[+/+]) and -deficient (Cav2.3[-/-]) mice using retinal vessel analysis. Methods: The mice were anesthetized and the pupil of one eye was dilated by application of a mydriaticum. An adapted prototype of retinal vessel analyzer was used to perform dynamic retinal vessel analysis. Arterial and venous responses were quantified in terms of the area under the curve (AUCart/AUCven) during flicker application, mean maximum dilation (mMDart/mMDven) and time to maximum dilation (tMDart/tMDven) during the flicker, dilation at flicker cessation (DFCart/DFCven), mean maximum constriction (mMCart/mMCven), time to maximum constriction (tMCart/tMCven) after the flicker and reactive magnitude (RMart/RMven). Results: A total of 33 retinal scans were conducted in 22 Cav2.3[+/+] and 11 Cav2.3[-/-] mice. Cav2.3[-/-] mice were characterized by attenuated and partially reversed arterial and venous responses, as reflected in significantly lower AUCart (p = 0.031) and AUCven (p = 0.047), a trend toward reduced DFCart (p = 0.100), DFCven (p = 0.100), mMDven (p = 0.075), and RMart (p = 0.090) and a trend toward increased tMDart (p = 0.096). Conclusion: To our knowledge, this is the first study using a novel, non-invasive analysis technique to document impairment of retinal vessel responses in VGCC-deficient mice. We propose that Cav2.3 channels could be involved in NVC and may contribute to the impairment of vasomotor responses under pathophysiological conditions.

Elevating intracranial pressure reverses the decrease in deoxygenated hemoglobin and abolishes the post-stimulus overshoot upon somatosensory activation in rats.

BOLD fMRI localizes activated brain areas by measuring decreases of deoxygenated hemoglobin (deoxy-Hb) caused by neurovascular coupling. To date, it is unclear whether intracranial pressure (ICP) modifies deoxy-Hb signaling for brain mapping. In addition, ICP elevation can test whether the BOLD post-stimulus undershoot, a transient hypo-oxygenation following functional activation, is due to vascular compliance rather than elevated cerebral metabolic rate of oxygen (CMRO(2)). We addressed these questions by studying the effect of ICP elevation on neurovascular coupling. In anesthetized rats, a cranial window was implanted over the somatosensory cortex. Using laser Doppler flowmetry and optical spectroscopy, changes in cerebral blood flow (CBF), cerebral blood volume (CBV) and deoxy-Hb were measured during electrical forepaw stimulation. Neuronal activity was monitored by somatosensory evoked potentials. ICP was elevated by subarachnoideal and intracisternal infusion of artificial cerebrospinal fluid. ICP elevation did not abrogate neurovascular coupling. However, the concomitant deoxy-Hb decrease was reduced (ICP=14mmHg) and reversed (ICP=28mmHg). Therefore, the validity of BOLD fMRI has to be questioned during increased ICP. Moreover, the amplitude of the deoxy-Hb post-stimulus overshoot was reduced with ICP elevation. CMRO(2) was not elevated during the post-stimulus response. Therefore, these data provide experimental evidence that the BOLD post-stimulus undershoot is a passive vascular phenomenon.

Acute changes of pro-inflammatory markers and corticosterone in experimental subarachnoid haemorrhage: A prerequisite for severity assessment.

Many details of the pathophysiology of subarachnoid haemorrhage (SAH) still remain unknown, making animal experiments an indispensable tool for assessment of diagnostics and therapy. For animal protection and project authorization, one needs objective measures to evaluate the severity and burden in each model. Corticosterone is described as a sensitive stress parameter reflecting the acute burden, and inflammatory markers can be used for assessment of the extent of the brain lesion. However, the brain lesion itself may activate the hypothalamic-pituitary-adrenal-axis early after SAH, as shown for ischemic stroke, probably interfering with early inflammatory processes, thus complicating the assessment of severity and burden on the basis of corticosterone and inflammation. To assess the suitability of these markers in SAH, we evaluated the courses of corticosterone, IL-6 and TNF-α up to 6h in an acute model simulating SAH in continuously anaesthetized rats, lacking the pain and stress induced impact on these parameters. Animals were randomly allocated to sham or SAH. SAH was induced by cisterna magna blood-injection, and intracranial pressure and cerebral blood flow were measured under continuous isoflurane/fentanyl anaesthesia. Withdrawn at predetermined time points, blood was analysed by commercial ELISA kits. After 6h the brain was removed for western blot analysis of IL-6 and TNF-α. Serum corticosterone levels were low with no significant difference between sham and SAH. No activation of the HPA-axis was detectable, rendering corticosterone a potentially useful parameter for stress assessment in future chronic studies. Blood IL-6 and TNF-α increased in both groups over time, with IL-6 increasing significantly more in SAH compared to sham towards the end of the observation period. In the basal cortex, IL-6 and TNF-α increased only in SAH. The pro-inflammatory response seems to start locally in the brain, reflected by an increase in peripheral blood. An additional surgery-induced systemic inflammatory response should be considered.

Assessment of cerebral hemodynamics during neurosurgical procedures using laser speckle image analysis.

Aneurysmal subarachnoid hemorrhage (aSAH) is a severe medical condition associated with a significant cause of mortality throughout the world. Cisterna magna injection model is accepted widely to mimic clinical aSAH and is performed on small animal models to study aSAH during neurosurgery. Coherent light scattered from the surface of the rat brain is used to infer information about the variations in blood flow during this condition. We obtained speckle images from the exposed cortex during the entire experiment using an external tissue imaging system. Contrast and fractal analyses are carried out for the recorded speckle pattern time series. Correlation analysis based on Hurst exponent for these images is found to be a more sensitive tool in studying aSAH as compared to routinely used laser speckle contrast analysis for assessing the changes in blood flow velocity. Additionally, our studies provide improved blood flow detection sensitivity with image Hurst exponent in combination with computed fractal dimension, during an event of aSAH.

Perspective review of optical imaging in welfare assessment in animal-based research.

To refine animal research, vital signs, activity, stress, and pain must be monitored. In chronic studies, some measures can be assessed using telemetry sensors. Although this methodology provides high-precision data, an initial surgery for device implantation is necessary, potentially leading to stress, wound infections, and restriction of motion. Recently, camera systems have been adapted for animal research. We give an overview of parameters that can be assessed using imaging in the visible, near-infrared, and thermal spectrum of light. It focuses on heart activity, respiration, oxygen saturation, and motion, as well as on wound analysis. For each parameter, we offer recommendations on the minimum technical requirements of appropriate systems, regions of interest, and light conditions, among others. In general, these systems demonstrate great performance. For heart and respiratory rate, the error was <4 beats / min and 5 breaths/min. Furthermore, the systems are capable of tracking animals during different behavioral tasks. Finally, studies indicate that inhomogeneous temperature distribution around wounds might be an indicator of (pending) infections. In sum, camera-based techniques have several applications in animal research. As vital parameters are currently only assessed in sedated animals, the next step should be the integration of these modalities in home-cage monitoring.

The Acute Phase of Experimental Subarachnoid Hemorrhage: Intracranial Pressure Dynamics and Their Effect on Cerebral Blood Flow and Autoregulation.

Clinical presentation and neurological outcome in subarachnoid hemorrhage (SAH) is highly variable. Aneurysmal SAH (aSAH) is hallmarked by sudden increase of intracranial pressure (ICP) and acute hypoperfusion contributing to early brain injury (EBI) and worse outcome, while milder or non-aneurysmal SAH with comparable amount of blood are associated with better neurological outcome, possibly due to less dramatic changes in ICP. Acute pressure dynamics may therefore be an important pathophysiological aspect determining neurological complications and outcome. We investigated the influence of ICP variability on acute changes after SAH by modulating injection velocity and composition in an experimental model of SAH. Five hundred microliters of arterial blood (AB) or normal saline (NS) were injected intracisternally over 1 (AB1, NS1), 10 (AB10, NS10), or 30 min (AB30) with monitoring for 6 h (n = 68). Rapid blood injection resulted in highest ICP peaks (AB1 median 142.7 mmHg [1.Q 116.7-3.Q 230.6], AB30 33.42 mmHg [18.8-38.3], p < 0.001) and most severe hypoperfusion (AB1 16.6% [11.3-30.6], AB30 44.2% [34.8-59.8]; p < 0.05). However, after 30 min, all blood groups showed comparable ICP elevation and prolonged hypoperfusion. Cerebral autoregulation was disrupted initially due to the immediate ICP increase in all groups except NS10; only AB1, however, resulted in sustained impairment of autoregulation, as well as early neuronal cell loss. Rapidity and composition of hemorrhage resulted in characteristic hyperacute hemodynamic changes, with comparable hypoperfusion despite different ICP ranges. Only rapid ICP increase was associated with pronounced and early, but sustained disruption of cerebral autoregulation, possibly contributing to EBI.

Intravenous rosuvastatin for acute stroke treatment: an animal study.

BACKGROUND AND PURPOSE: Statins exert rapid cholesterol-independent vasoprotective effects. Here, we tested whether postevent treatment with intravenously (i.v.) administered rosuvastatin improves acute stroke outcome in mice. METHODS: 129/SV wild-type mice were subjected to 1-hour filamentous middle cerebral artery occlusion (MCAo), followed by reperfusion, and were postevent treated with i.v. or intraperitoneal (i.p.) rosuvastatin given up to 6 hours after MCAo (dose range 0.02 to 20 mg kg(-1) body weight). RESULTS: Rosuvastatin, when administered i.v., significantly reduced lesion size when given up to 4 hours after MCAo and in doses as low as 0.2 mg kg(-1). In contrast, i.p. administration provided protection only when given directly on reperfusion at a dose of 20 mg kg(-1) but not at lower doses or later time points. Lesion protection was evident as late as 5 days after brain ischemia and was associated with functional improvements in the pole-test and wire-hanging test (2.0 mg kg(-1) dose). Neuroprotection with i.v. rosuvastatin was achieved with peak plasma concentrations <0.5 ng ml(-1) (ie, with 0.2 mg kg(-1)) and was associated with increased levels of phosphorylated Akt kinase and endothelial nitric oxide synthase in the vasculature. CONCLUSIONS: Rosuvastatin, given intravenously at pharmacologically relevant concentrations, protects from focal brain ischemia up to 4 hours after an event. In our opinion, the development of an intravenous statin formulation is warranted for acute stroke trials with statins in humans.

In vivo imaging of the inflammatory receptor CD40 after cerebral ischemia using a fluorescent antibody.

BACKGROUND AND PURPOSE: Brain inflammation is a hallmark of stroke, where it has been implicated in tissue damage as well as in repair. Imaging technologies that specifically visualize these processes are highly desirable. In this study, we explored whether the inflammatory receptor CD40 can be noninvasively and specifically visualized in mice after cerebral ischemia using a fluorescent monoclonal antibody, which we labeled with the near-infrared fluorescence dye Cy5.5 (Cy5.5-CD40MAb). METHODS: Wild-type and CD40-deficient mice were subjected to transient middle cerebral artery occlusion. Mice were either intravenously injected with Cy5.5-CD40MAb or control Cy5.5-IgGMAb. Noninvasive and ex vivo near-infrared fluorescence imaging was performed after injection of the compounds. Probe distribution and specificity was further assessed with single-plane illumination microscopy, immunohistochemistry, and confocal microscopy. RESULTS: Significantly higher fluorescence intensities over the stroke-affected hemisphere, compared to the contralateral side, were only detected noninvasively in wild-type mice that received Cy5.5-CD40MAb, but not in CD40-deficient mice injected with Cy5.5-CD40MAb or in wild-type mice that were injected with Cy5.5-IgGMAb. Ex vivo near-infrared fluorescence showed an intense fluorescence within the ischemic territory only in wild-type mice injected with Cy5.5-CD40MAb. In the brains of these mice, single-plane illumination microscopy demonstrated vascular and parenchymal distribution, and confocal microscopy revealed a partial colocalization of parenchymal fluorescence from the injected Cy5.5-CD40MAb with activated microglia and blood-derived cells in the ischemic region. CONCLUSIONS: The study demonstrates that a CD40-targeted fluorescent antibody enables specific noninvasive detection of the inflammatory receptor CD40 after cerebral ischemia using optical techniques.