1. the rat entorhinal cortex Croatian Science Foundation
Protocol for stereotaxic delivery of tau oligomers and tau fibrils into
the rat entorhinal cortex
Animals: the experiment was performed on nontransgenic rat model
It has been shown that the locus coeruleus is among the first structures affected by tau protein abnormalities during the course of Alzheimers’s disease
Progressive accumulation of misfolded tau protein in the central nervous system is one of the most predominant characteristic of taupathies.
the tau pathology manifests in early stages of the disease in locus coeruleus and enthorinal cortex, and progressivel spreads to other brain areas in a stereotypical fashion through anatomicall connected regions.
Croatian Science Foundation
Grant IP
( )
Lea Langer Horvat, Goran Šimić
Department of Neuroscience, Croatian Institute for Brain Research
School of Medicine, University of Zagreb, Šalata 12, Zagreb, Croatia
Introduction
Aim
Sporadic Alzheimer’s disease (AD) is the most common secondary tauopathy characterized by progressive loss of cognitive functions and behavioral impairment.
It has been shown that tau pathology progressively spreads in an anatomically stereotypical manner throughout the brain with the first lesions in the locus coeruleus and the entorhinal cortex from where they spread to the hippocampus and high-order neocortical regions.
Recent studies on transgenic animal models of AD provide initial evidence that pathogenic protein aggregates could propagate from neuron to neuron, and then self-replicate by templated misfolding of monomeric tau proteins thus igniting further spreading.
In this protocol, we describe stereotaxic surgery optimized for delivery of tau oligomers and tau fibrils into the rat entorhinal cortex to assess whether such an intervention will cause a neuron-to-neuron physical spread of tau oligomers and tau aggregates from the site of injection to the entorhinal projecting cortical regions and and will those changes be associated with expected cognitive impairment.
Adopted from Šimić et.al, 2017
Adopted from Walsh and Selkoe, 2016
Materials and methods
The study will include 3-4 months old male Wistar rats (n=108) divided into three groups.
Rats from the first experimental group will be stereotaxically injected with tau oligomers (4 μg) into the entorhinal cortex, the second group will be injected with preformed tau fibrils (4 μg) and the third, control group, with phosphate-buffered saline.
Animals will be tested and analyzed 3 days (6 rats per group), 3 months, 6 months and 9 months post-injection (10 rats per group).
Validation of coordinates for the entorhinal cortex (EC)
Figure 1. Verification of coordinates for the entorhinal cortex (EC) in the rat using cresyl violet
1. Stereotaxic coordinates for the entorhinal cortex determined from the 2004 stereotaxic rat brain atlas of Paxinos and Watson
108 male Wistar rats (age 3-4 months)
trace of cresyl-violet
group injected with tau oligomers
group injected with tau fibrils
group injected with PBS
2. For targeting the medial entorhinal cortex it is necessary to use a 10°inclination of the microinjection unit in mediolateral direction
Validated coordinates AP mm
ML 3.30 mm
DV 8.63 mm
Inclination 10°
Key technical requirements had to be fulfilled before and during the procedure so that the target structure is attained with maximal precision. The stereotaxic coordinates need to be validated relative to age, sex, strain, and the topography of the vascular system of the rat.
Adopted from Paxinos and Watson, 2004
Expected scientific contribution
Acknowledgments
This work is funded by the Croatian Science Foundation HRZZ (IP ) and by the European Union through the European Regional Development Fund, Operational Programme Competitiveness and Cohesion, grant agreement no. KK , CoRE – Neuro.
We hope that our approach will improve the understanding of mechanisms in spreading of the pathological changes in AD brain and to develop a model that will mimic early events underlying those changes in human disease. The ultimate goal would be to define possible new therapeutic targets and interventions aimed at blocking tau protein oligomerization, aggregation, and spread.
References: Šimić G, Babić Leko M, Wray S, Harrington CR, Delalle I, et al Monoaminergic neuropathology in Alzheimer’s disease. Prog. Neurobiol. 2017;151:101–138.; Peeraer E, Bottelbergs A, Van Kolen K, Stancu I-C, Vasconcelos B, Mahieu M, et al. Intracerebral injection of preformed synthetic tau fibrils initiates widespread tauopathy and neuronal loss in the brains of tau transgenic mice. Neurobiol Dis. 2015;73:83–95.; Gerson JE, Sengupta U, Kayed R. Tau oligomers as pathogenic seeds: preparation and propagation in vitro and in vivo. Methods Mol. Biol. 2017;1523:141–57.