LAUSR

Introduction/Hypothesis: Cerebellar abnormalities, particularly those related to cerebellar hemorrhage (CBH), have become increasingly associated with preterm birth and adverse neurodevelopmental outcomes. The mechanism by which CBH affects the developing cerebellum is largely unknown. Our objectives were to develop a novel murine model of preterm posterior fossa subarachnoid hemorrhage, determine the effect of CBH on cerebellar development, and investigate neuroinflammation as a mechanism of injury. Methods: In this study, 20 μl of whole blood or artificial cerebrospinal fluid (aCSF) was injected into the posterior fossa of post-natal day (P)6 CD1 mice (n=8/group). Animals in both blood and aCSF groups were administered either ketoprofen (n=4/group) or buprenorphine (n=4/group). At P8, cerebellar tissue was analyzed by immunohistochemistry targeting BrDU (cell proliferation and migration), Ki-67 (cell proliferation), Caspase-3 (cell death), GFAP (Bergman glia), and Calbindin (Purkinje cells). Results: CBH resulted in significantly decreased external granule layer (EGL) thickness and Purkinje cell (PC) density compared to the aCSF group (22.98 μm ± 6.489 vs. 34.08 μm ± 4.317, p= 0.001; 6.625 ± 1.506 cells/200 μm vs. 9.250 ± 1.035 cells/200 μm, p= 0.0052, respectively). Bergmann glial (BG) fiber crossings were significantly increased in the CBH group compared to those in the aCSF group (24.83 ± 3.486 crossings/200 μm vs. 20.42 ± 3.528 crossings/200 μm, p= 0.013). BG fibers in the CBH group demonstrated irregular morphology and end plate retraction. Treatment with ketoprofen did not have a significant effect on EGL thickness, PC density, or BG fiber crossings/morphology. Conclusions: Our results demonstrate that CBH results in significant injury and derangement of cerebellar developmental programs in neonatal mice. Hemorrhagic injury is associated with significantly decreased PC density, thinning of the EGL, and increased BG fiber crossing. Altered BG morphology may be indicative of reactive gliosis. Treatment with ketoprofen did not attenuate the injury response, suggesting neuroinflammation is not the primary mechanism of injury. We postulate that CBH damages the PC layer, decreasing sonic hedgehog signaling and altering proliferation/differentiation in the EGL.