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."

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.

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.

Severity assessment and scoring for neurosurgical models in rodents.

The most important acute neurological diseases seen at neurosurgery departments are traumatic brain injuries (TBI) and subarachnoid hemorrhages (SAH). In both diseases the pathophysiological sequela are complex and have not been fully understood up to now, and rodent models using rats and mice are most suitable for the investigation of the pathophysiological details. In both models, surgery is performed under anesthesia, followed by assessment of their functional outcome and behavioral testing before brain tissue analysis after euthanasia. Postoperative analgesia is mandatory, and supplementary care is highly recommended for refinement purposes. Pain and stress assessment is mainly based on clinical and behavioral signs, and further research is needed to improve the evaluation of severity in these models.

Lipopolysaccharide inhibits ghrelin-excited neurons of the arcuate nucleus and reduces food intake via central nitric oxide signaling.

Lipopolysaccharide (LPS) induces anorexia and expression of inducible nitric oxide synthase (iNOS) in the hypothalamic arcuate nucleus (Arc). Peripheral administration of the iNOS inhibitor 1400 W counteracts the anorectic effects of LPS. Here we investigated the role of central NO signaling in LPS anorexia. In electrophysiological studies we tested whether 1400 W counteracts the iNOS-dependent inhibition of Arc neurons triggered by in vivo or in vitro stimulation with LPS. We used the hormone ghrelin as a functional reference stimulus because ghrelin is known to activate orexigenic Arc neurons. Further, we investigated whether in vitro LPS stimulation induces an iNOS-mediated formation of the second messenger cGMP. Since the STAT1 pathway contributes to the regulation of iNOS expression we investigated whether LPS treatment induces STAT1 phosphorylation in the Arc. Finally we tested the effect of intracerebroventricular injection of 1400 W on LPS-induced anorexia. Superfusion with 1400 W (10(-4) M) increased neuronal activity in 37% of neurons in Arc slices from LPS treated (100 µg/kg ip) but not from saline treated rats. Similarly, 1400 W excited 45% of Arc neurons after in vitro stimulation with LPS (100 ng/ml). In both approaches, a considerable percentage of 1400 W sensitive neurons were excited by ghrelin (10(-8)M; 50% and 75%, respectively). In vitro stimulation with LPS induced cGMP formation in the Arc, which was blocked by co-incubation with 1400 W. LPS treatment elicited a pSTAT1 response in the Arc of mice. Central 1400 W injection (4 µg/rat) attenuated LPS-induced anorexia and counteracted the LPS-dependent decrease in respiratory quotient and energy expenditure. In conclusion, the current findings substantiate a role of central iNOS dependent NO formation in LPS-induced effects on eating and energy homeostasis. A pharmacological blockade of NO formation might be a therapeutic approach to ameliorate disease-related anorexia.