P: 67 Hippocampal and Cerebellar Astrocytes Morphological Alterations in a Rat Model of Chronic Hepatic Encephalopathy

Abstract

BACKGROUND: Chronic hepatic encephalopathy (CHE) is a spectrum of neuropsychiatric abnormalities in patients with chronic liver disease. The hippocampus and cerebellum are key regions implicated in the cognitive and fine motor deficits of CHE.1 Astrocytes can sense neuronal activity through neurotransmitter-receptors and ion-channels, modulate the neural circuits and control energy homeostasis through morphological plasticity. They adjust their volume by releasing osmolytes (inositol, taurine, creatine) and can remodel their processes.2–4 They initiate synaptic development and regulate synaptic plasticity in both the healthy and injured brain.5 Astrocytes convert the neurotoxin ammonia into glutamine, regulate cerebral hemodynamics and cytokine responses to inflammation-related signaling pathways.3,6 Histological studies of BDL rat brains are controversial to date, some reporting no change, increased or decreased of anti-glial-fibrillary-acidic-protein (GFAP).Our aim was to investigate whether and how astrocytes react in the hippocampus and cerebellum of bile-duct-ligated (BDL) rat. METHODS: BDL-rats and sham-rats at 4 and 8-weeks post-BDL (n = 3/group/2-time-points) were anesthetized with 4% isoflurane and Temgesic (ESSEX) 0.1mg/kg before transcardial PBS perfusion. Brains were fixed in 4%-formaldehyde and cryopreserved in 30%-sucrose, embedded in a Tissue-Tek®OCT. Immunohistochemistry: On 16 micron sagittal-sections, GFAP7 and DAPI (nucleus) were used. For each rat (n = 3 at 4-weeks and n = 3 at 8-weeks post-BDL, n = 3 Shams), seven slides/rat were analyzed (distance between sections ∼250 microns). Morphometric measurements were performed using Sholl-analysis8 (∼1000 processes/group/region) (Figure 1c). RESULTS: Astrocytes activation is represented by the significant increase in GFAP+ cells at week4 post-BDL in the hippocampus (+47.5%) and cerebellum (+48.7%) vs SHAM (Figure 1a,b). Decrease in the processes intersection was observed already at week4 post-BDL (Figure 1e). Although at week8 post-BDL a significant reduction of astrocytes number was observed (∼20%) (Figure 1b) the astrocytes were altered morphologically, showing shortening (hippocampus: week 4 −13.3%, week 8 −32.4%; cerebellum: week 4: −17.3%, week 8: −35.5%) and decreased number of processes (hippocampus: week 4:−5.8%, week 8:−18.77%; cerebellum: week 4:−32.2%, week 8:−41.5%) and processes intersections at week 8 post−BDL (hippocampus: ring1: −14.5%, ring 2: −39%, ring 3: −72.5%; cerebellum: ring1: −17.6%, ring 2: −27.4%, ring 3: −58.7%) as well as minor processes thickening (Figure 1d). The reported % changes are relative to SHAM. CONCLUSIONS: To our knowledge, this is the first report showing significant alterations in astrocytes count and important morphological changes already 4-weeks post-BDL in the hippocampus and cerebellum. Increase in GFAP+ cells may be related to the stimulation of mature astrocytes and reentering into proliferation cycle which is similar to proliferating neonatal astrocytes and is a common situation in various neuropathological disorders.9,10 The proliferation of activated glial-cells can induce additional inflammatory reactions and creation of glial-scar, something to be investigated in future studies. Finally, the astrocytosis and astrocyte morphology changes may alter the CNS microenvironment that usually ensures neuronal health and may contribute to the cognitive impairment of BDL rats.