Inducing sterile pyramidal neuronal death in mice to model distinct aspects of gray matter encephalitis

Abstract

Up to one person in a population of 10,000 is diagnosed once in lifetime with an encephalitis, in 50–70% of unknown origin. Recognized causes amount to 20–50% viral infections. Approximately one third of affected subjects develops moderate and severe subsequent damage. Several neurotropic viruses can directly infect pyramidal neurons and induce neuronal death in cortex and hippocampus. The resulting encephalitic syndromes are frequently associated with cognitive deterioration and dementia, but involve numerous parallel and downstream cellular and molecular events that make the interpretation of direct consequences of sudden pyramidal neuronal loss difficult. This, however, would be pivotal for understanding how neuroinflammatory processes initiate the development of neurodegeneration, and thus for targeted prophylactic and therapeutic interventions. Here we utilized adult male NexCreERT2xRosa26-eGFP-DTA (= ‘DTA’) mice for the induction of a sterile encephalitis by diphtheria toxin-mediated ablation of cortical and hippocampal pyramidal neurons which also recruits immune cells into gray matter. We report multifaceted aftereffects of this defined process, including the expected pathology of classical hippocampal behaviors, evaluated in Morris water maze, but also of (pre)frontal circuit function, assessed by prepulse inhibition. Importantly, we modelled in encephalitis mice novel translationally relevant sequelae, namely altered social interaction/cognition, accompanied by compromised thermoreaction to social stimuli as convenient readout of parallel autonomic nervous system (dys)function. High resolution magnetic resonance imaging disclosed distinct abnormalities in brain dimensions, including cortical and hippocampal layering, as well as of cerebral blood flow and volume. Fluorescent tracer injection, immunohistochemistry and brain flow cytometry revealed persistent blood–brain-barrier perturbance and chronic brain inflammation. Surprisingly, blood flow cytometry showed no abnormalities in circulating major immune cell subsets and plasma high-mobility group box 1 (HMGB1) as proinflammatory marker remained unchanged. The present experimental work, analyzing multidimensional outcomes of direct pyramidal neuronal loss, will open new avenues for urgently needed encephalitis research.

NMDAR1 autoantibodies amplify behavioral phenotypes of genetic white matter inflammation: a mild encephalitis model with neuropsychiatric relevance

“…2F, G ), consistent with a clear BBB disruption, allowing NMDAR1-AB to readily reach the brain. Similar to the observations in gray matter encephalitis after diphtheria toxin-induction [ 51 ], the complete lack of peripheral changes in a situation of substantial white matter inflammation, including BBB breakdown and inflammatory degeneration, is intriguing. At the same time it is alarming for clinicians who are not infrequently confronted with the necessity to diagnose or exclude an encephalitis in vivo, particularly upon suddenly occurring behavioral abnormalities in neuropsychiatric practice [ 51 ].…”

“… A Fluorojade C staining as sensitive marker of dying neurons shows absence of neuronal death in all three groups; representative images provided; as positive control image, a section of a mouse after induced pyramidal neuronal death is given [ 51 ]. ( B , C ) Representative images of microglia and astrocytes in corpus callosum ( B ) and hippocampal CA1 region ( C ), depicting prominent reactive gliosis in corpus callosum of Cnp −/− mice, and virtually normal astrocytes and ramified microglia in the hippocampus; images acquired as 10 µm Z-stacks, displayed as maximum-intensity projections.…”

“…To assess hippocampus-dependent cognitive functions such as spatial learning and memory, we conducted the classical Morris water maze (MWM) as previously described [ 49 – 51 ]. Immunized mice were tested at 50–64 days post-immunization (DPI); cKO mice at the age of 3 months, including a subsequent reversal learning period followed by a second probe trial.…”

“…Fixed hemispheres were cut into 30 µm coronal sections on a cryostat (CM1950, Leica, Wetzlar, Germany) and stored at −20 °C in cryomedium (25% ethylene glycol/25% glycerol/PBS). Quantifications were performed as described previously [ 24 , 51 ], using 3–5 regularly spaced sections per mouse (every 150 µm) between Bregma coordinates −1.34 and −2.24 mm. Free-floating frozen sections were blocked and permeabilized for 1 h at RT with 5% normal horse serum (NHS, 26050-088, Thermo) in 0.5% Triton X-100/PBS and incubated overnight at 4 °C with primary antibodies.…”

“…Data from 3–5 hippocampi/mouse was averaged. Fluorojade C staining of dying neurons was performed as previously described [ 51 ]. Neuronal death was qualitatively evaluated in hippocampus and corpus callosum by a blinded investigator using two sections per mouse.…”