engleski

Protocol for stereotaxic delivery of tau monomers and oligomers into the rodent brain

Introduction: This protocol describes stereotaxic surgery for the delivery of different tau species in the rat brain. The surgery is optimized to result in minimal injury to the brain parenchyma and to provide reproducible results for the stereotaxic targeting of specified brain regions, ventral entorhinal cortex (1 mm x 0, 8 mm) and locus coeruleus (0, 8 mm x 0, 3 mm). Crucial technical gestures have to be carried out before and during the procedure so that the target structure is attained with a maximum of precision. The stereotactic coordinates need to be validated relative to age, sex, strain and the cartography of the vascular system of the rat. Depending on the location of the target structure, the cerebrovascular anatomy of the animal can be a constraint in the insertion of microinjection cannulae, such as in targeting locus coeruleus.Usual for a structure located in anterior part of the brain, bregma is recommended as a landmark. On the other hand, if the target is the entorhinal cortex or locus coeruleus, structures located in the posterior brain, it is more judicious to use lambda as the reference point. Methodology: For stereotaxic surgery, we used 12 months old male and female Lewis rats. Stereotaxic coordinates for locus coeruleus and entorhinal cortex are determined from stereotaxic atlas (George Paxinos and Charles Watson, Academic Press, 2004) and optimized according to age and sex of the rat using cresyl violet tracer. To determine if selected stereotaxic coordinates for entorhinal cortex and locus coeruleus are associated with brain regions remote from the injected area and anatomically connected through well-defined neural network anatomical, tracers are used.Macroscopic observation of serial frozen sections at the level of the marked zone allows a validation of and the possibility to correct, if needed, the stereotaxic coordinates of the target structure. This is especially useful when the target region is very small, such as locus coeruleus. Results: Data collection is still in progress. Conclusion: For calculating stereotaxic coordinates of the target structure it is important to take into account the organization of the cerebral vascular system of the animal. To target a structure such as the nucleus locus coeruleus, which is close to the midline axis, it is necessary to use an angle due to the presence of the transverse venous sinus. The obstacle that this blood vessel presents can be bypassed by a forward inclination of the head at an angle of around 15°. For targeting ventral entorhinal cortex, which is not in the arbitrary work surface of the skull, it is necessary to use a 14° inclination of the microinjection unit. One must be vigilant about the starting point of the descent of the cannula, since there can be a difference of up to 1 mm between determination of the dorso- ventral coordinate either from the surface of the skull or from the surface of the dura mater. A very rigorous approach is necessary for the correct determination of the stereotaxic coordinates.

stereotaxic surgery ; locus coeruleus ; entorhinal cortex

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