Cerebrospinal fluid dynamics disorders after subarachnoid haemorrhage in an experiment.

Abstract

Havryliv T.S. Cerebrospinal fluid dynamics disorders after subarachnoid haemorrhage in an experiment. – Qualifying scientific work in manuscript form. Dissertation for the degree of Doctor of Philosophy in the specialty 222 "Medicine" (22 – Health Care). – State University "Uzhhorod National University", Ministry of Education and Science of Ukraine, Uzhhorod, 2026. According to the latest complete global data from the World Health Organization (2021), stroke ranked as the third leading cause of death worldwide, following ischemic heart disease and COVID-19. Subarachnoid hemorrhage (SAH), a subtype of hemorrhagic stroke, leads to the development of communicating post-hemorrhagic hydrocephalus (CPHH) in approximately one-third of patients. Up to 45% of these patients require permanent cerebrospinal fluid diversion, while nearly half of them experience shunt dysfunction within the first year of follow-up. One key mechanism underlying CPHH development is the accumulation of hemoglobin degradation products, which induce subarachnoid fibrosis and impair cerebrospinal fluid resorption. Iron plays a crucial role in these processes, as its excess contributes to neuroinflammation and secondary damage to brain structures. In this context, the use of iron-chelating agents is considered a promising approach to mitigating iron-induced injury following SAH. This dissertation aimed to test the hypothesis that iron-chelating therapy reduces the development of communicating post-hemorrhagic hydrocephalus in an experimental model of SAH. The study was conducted using Wistar rats weighing 250-500 g. Animals were divided into four groups. The control group underwent no surgical procedures. In the sham group, 0.15 mL of isotonic saline was injected into the cisterna magna, followed by a second injection 48 hours later. In the third group (blood group minus minocycline, BGMM), 0.15 mL of autologous blood was injected into the cisterna magna, with a repeated injection administered after 48 hours. The fourth group (blood 7 group plus minocycline, BGPM) underwent the same hemorrhagic protocol and received the iron-chelating agent minocycline. Transcranial ultrasonography was performed in all groups at predefined time points. In the control group, examinations were conducted according to the same schedule without surgical intervention, whereas in the experimental groups, ultrasonography was performed at baseline and during postoperative follow-up. Ventricular size was assessed using the Levene index (LI). CPHH was defined as a ventricular index exceeding the 97th percentile of the pre-intervention LI (1.297). Morphological features of SAH, including subarachnoid blood accumulation and ventricular wall damage, were evaluated histologically. During the study, а total of 97 experimental interventions were performed in 50 rats. The overall mortality rate was 12%, while mortality was higher in the hemorrhagic groups, reaching 15%. According to ultrasonographic assessment, hydrocephalus developed in 56% of experimental animals in the hemorrhagic group that did not receive the iron-chelating agent. In animals of this group, histological examination revealed subarachnoid and intraventricular hemorrhage, accompanied by disruption of the ependymal lining, findings consistent with ultrasonographic evaluation. Administration of minocycline in the BGPM group prevented ventricular enlargement following autologous blood injection, as evidenced by comparable preoperative and postoperative Levene index values (1.079 ± 0.096 and 1.034 ± 0.058, respectively). The difference between the BGMM and BGPM groups was highly significant (mean difference 0.179 ± 0.029, t(41) = 6.12, p < 0.00001). The results indicate that minocycline significantly reduces the risk of post- hemorrhagic ventriculomegaly in experimental animals. These findings suggest that iron-chelating agents represent a promising therapeutic approach for the prevention and treatment of post-hemorrhagic hydrocephalus in clinical practice. The scientific novelty of the obtained results. 1. This study substantiates the effectiveness of an improved double-hemorrhage experimental model of subarachnoid hemorrhage, incorporating interval-based administration of autologous blood into the cisterna magna, for reproducing 8 communicating posthemorrhagic hydrocephalus under conditions of low postoperative mortality. 2. The study expands current knowledge on the patterns of cerebrospinal fluid dynamic disturbances in experimental repeated subarachnoid hemorrhage, reproducing the key features of the clinical course of posthemorrhagic hydrocephalus in humans. 3. The study clarifies the pathogenetic significance of morphological brain changes following subarachnoid hemorrhage, particularly damage to the ependymal lining of the ventricles and intraventricular blood accumulation, in the 4. development of impaired cerebrospinal fluid resorption. The diagnostic value of transcranial ultrasonography is evaluated as a dynamic, quantitative method for assessing ventriculomegaly in small laboratory animals, based on comparison with morphological findings. 5. The study experimentally demonstrates the preventive effect of the iron- chelating drug minocycline on the development of posthemorrhagic ventriculomegaly, with a statistically significant reduction in its incidence in experimental animals. Practical significance of the obtained results. 1. 2. 3. Prophylactic use of iron-chelating agents may potentially reduce the risk of post- hemorrhagic hydrocephalus in patients with subarachnoid hemorrhage, thereby decreasing the need for additional neurosurgical interventions, including ventriculoperitoneal shunting and endoscopic third ventriculostomy. The applied double-hemorrhage experimental model, involving interval injection of autologous blood into the cisterna magna, is a reliable and effective method for reproducing severe cerebrovascular pathology with clinically relevant analogues in humans and may be used in further preclinical studies. Non-invasive ultrasonographic examination of the brain is a valid and informative method for diagnosing ventriculomegaly in small laboratory animals, allowing real-time dynamic assessment and making it suitable for serial experimental studies. 9 Experimental research was conducted at the Lithuanian University of Health Sciences using equipment located at the Biological Research Center in Kaunas, Lithuania. All experimental procedures involving animals were performed in accordance with the Republic of Lithuania Law on the care, keeping, and use of experimental vertebrate animals and Directive 2010/63/EU of the European Parliament. The study was approved by the State Food and Veterinary Service of Lithuania (No. G2-126, October 29, 2019). The author holds a certificate from the Federation of European Laboratory Animal Science Associations (FELASA) and the Society for Laboratory Animal Science (SOLAS), category B, issued by Humboldt University of Berlin / Berlin Mouse Clinic for Neurology and Psychiatry. Compliance with international and national standards for biomedical research was approved by the Bioethics Commission of Uzhhorod National University (Protocol No. 2/16, dated October 4, 2022). The results of the dissertation research have been introduced into the scientific and methodological work of the Department of Neurology, Psychiatry and Neurosurgery at Uzhhorod National University.