Why neonatal and adult strokes are different Why neonatal and adult strokes are different? The role of blood-brain barrier and cerebral blood flow Sindeeva Olga, Saratov State University, Russia Oxana Semyachkina-Glushkovskaya, Saratov State University, Russia Tuchin Valery Victorovich, Saratov State University SFM 2015

Neonatal and adult strokes Recently, it has been believed that stroke is common only for adults. However, due to the progress in neuroimaging, it has became obvious that the incidence of stroke in newborns is similar to adults. The neonates have higher risk of stroke but lower mortality and more favorable outcome than adults.

The differences between adult and neonatal strokes 50% ischemic, 50% hemorrhagic stroke 87% ischemic, 6.5% - 13% hemorrhagic stroke Newborns Adults Traditional Risk Factors Physiological or traumatic factors:  Anomalies of brain vessels Medical disorders Lifestyle factors:  Hypertension Smoking Chronic alcohol Use of medications (anticoagulants, sympathomimetic agents) Stress Common Hemorrhage Location The cortex and subcortical tissues (full term neonates)  The germinal matrix (preterm neonates) The region of the middle cerebral artery Typical Clinical Presentation Usually without any outward clinical symptoms or with non-specific neurological signs Loss of consciousness Coma Weakness of the opposite side of the body Affected language, sensory and motoric function

New model of brain hemorrhage in newborn rats To induce of brain bleeding we used sound stress (17 Hz and 120 dB) during 2 hours

Model of brain hemorrhage in adult rats Adult rats more resistant to the damaging effects of stress sound We implanted a silver clip on the left renal artery 7 weeks after surgery in adult rats develop hypertension To induce of brain bleeding in adult hypertensive rats we used sound stress (17 Hz and 120 dB) during 2 hours

Age-related differences in development of hemorrhagic stroke in newborn and adult rats using histological analysis newborn rat adult rat The typical example of size of stress-induced hemorrhages in the brain of newborn (on the left) and adult (on the right) rats assessed by histological methods. Hematoxylin & Eosin staining. Using histological method as a gold standard for the morphological evaluation of pathological changes in tissues, we found that sound stress induced more severe hemorrhages in adult rats compared with newborn rats. Indeed, the size of hemorrhages was larger in adult rats compared with newborn rats. Newborn rats demonstrated small diffuse bleedings in the cortex while adult rats had more severe bleedings in the brain parenchyma

Age-related differences in development of hemorrhagic stroke in newborn and adult rats using MRT analysis The typical example of common type of stress-induced hemorrhages in the brain of adult (on the top) and newborn (on the bottom) rats: A and C – MRI imaging of normal brain tissues; B and D - the intensive MRI signals show the hemorrhagic impregnation of the brain tissues; B – the hemorrhages in cerebellum (blue arrow) and cerebellar nucleus (red arrow); D – small hemorrhages in the brain cortex (arrowed). The adult and newborn rats demonstrated different types of hemorrhages in the different anatomical structures of the brain. Indeed, all newborn rats had small hemorrhages in the cortical and subcortical tissues. The adult rats were presented with hemorrhages in the cerebellum and cerebellar nucleus, we found only one case with the cortical bleeding that also was accompanied by cerebellar hemorrhage.

Photoacoustic imaging newborn rat adult rat Newborn rats with stroke showed increased vascular permeability to erythrocytes (diapedetic hemorrhage). Adult rats showed extensive bleeding from damaged vessels

The study of changes in diameter of the sagittal sinus after adrenaline injection before and after stroke in newborn and adult rats using DOCT Groups Before stroke After stroke Basal values Effect of adrenaline Newborn rats 0.30±0.03 0.21±0.02* 1.67±0.04† 1.65±0.07† Adult rats 0.33±0.01 0.23±0.03* 0.71±0.03 0.63±0.05* p<0.05 vs.: * - basal levels; † - age groups In normal condition, adrenaline infusion caused constriction of the sagittal sinus that was accompanied by decrease in diameter of this cerebral vein. There were no age differences in vascular effect of adrenaline. So, adrenaline induced the decrease in diameter of the sagittal sinus by 30% (p<0.05) in newborn rats and by 31% (p<0.05) in adult rats.

The changes in diameter of the sagittal sinus (%) after adrenaline injection in newborn and adult rats under normal state and during stroke development. The basal values of diameter of sagittal sinus (no adrenaline) were expressed as 100%. The stroke incidence was accompanied by complete loss of sensitivity of the sagittal sinus to adrenaline in newborn rats. The adult rats demonstrated tendency to suppression of adrenergic vasoconstrictor effect. The changes of normal vascular responses to adrenaline during the stroke development can be explained via the difficult vessel constriction due to the critical dilation of the sagittal sinus and accumulation of extensive blood in this vessel that was more pronounced in newborn than in adult animals. Other possible mechanism may lead in age differences in the BBB permeability.

The role of the blood-brain barrier (BBB) in the development of stroke The blood-brain barrier is an intricately coordinated mechanism for protecting the brain. The BBB is a cellular and metabolic barrier located at the capillaries in the brain that alters permeability, restricting the passage of some chemical substances and microscopic objects from the bloodstream into the neural tissue, while allowing other substances to pass into the brain. An important question remains, how changes the permeability of the BBB after stroke at different ages?

The color test for the study of age differences in the stroke-induced breakdown of BBB permeability with using Evans blue injection adult rat newborn rat The intravenous infusion of Evans Blue was not accompanied any changes in the brain tissues in both intact newborn and adult rats. But, the stroke development was associated with the leakage of Evans Blue into the brain the next day after dye administration in adult but not in newborn rats

The expression of two main structural and functional elements of the BBB – occludin and claudin-5 in newborn and adult rats The more significant expression of tight junction proteins of BBB (occludin and claudin-5) in newborns vs. adults is one of possible mechanisms underlying age differences in stroke-related changes in BBB permeability.

The expression of two main structural and functional elements of the BBB – occludin and claudin-5 in newborn and adult rats The immunohistochemistry study of expression of occludin and claudin-5 (brown color) in cerebral vessels (arrowed) in adult (on the left) and newborn (on the right) rats. The western blotting results were confirmed by immunohistochemistry data

The expression of claudin-5 in large cerebral veins in newborn and adult rats The immunohistochemistry study of expression of claudin-5 (brown color) in cerebral veins (arrowed) in newborn (on the left) and adult (on the right) rats. The expression of claudin-5 in large cerebral veins in newborn animals than in adult rats

Conclusions Using histological methods, MRT analysis and photoacoustic imaging of cerebral vessels geometry, we found age differences in type of stroke. Newborn rats demonstrated small multifocal diffuse bleedings in the cortex while adult rats had more severe bleedings in the brain parenchyma. Using Doppler optical coherent tomography, we found higher stress-reactivity of sagittal sinus (major cerebral vein) to deleterious effects of stress in newborn vs. adult rats suggesting that the cerebral veins are more vulnerable to negative factors in newborns than adults. The stress-induced stroke is accompanied by age differences in breakdown of brain blood barrier (BBB) permeability that was decreased in newborn rats and increased in adult rats. The more significant expression of tight junction proteins of BBB (occludin and claudin-5) in newborns vs. adults is one of possible mechanisms underlying age differences in stroke-related changes in BBB permeability and cerebral venous circulation.

Thanks for your attention! This work was supported by Grant of Russian Science Foundation (№ 14-15-00128).