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.

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.

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.”

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 seven 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. 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 (PCA) resulted in a clear separation of sham surgery and SAH animals from their respective baseline as well as from the anesthesia only group at day 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 non-neurological disease models.