Abstract
Translational research often requires the testing of experimental therapies in primates, but research in non-human primates is now stringently controlled by law around the world. Tissues fixed in formaldehyde without glutaraldehyde have been thought to be inappropriate for use in electron microscopic analysis, particularly those of the brain. Here we report the immunoelectron microscopic characterization of arginine vasopressin (AVP)-producing neurons in macaque hypothalamo-pituitary axis tissues fixed by perfusion with 4% formaldehyde and stored at −25 °C for several years (4–6 years). The size difference of dense-cored vesicles between magnocellular and parvocellular AVP neurons was detectable in their cell bodies and perivascular nerve endings located, respectively, in the posterior pituitary and median eminence. Furthermore, glutamate and the vesicular glutamate transporter 2 could be colocalized with AVP in perivascular nerve endings of both the posterior pituitary and the external layer of the median eminence, suggesting that both magnocellular and parvocellular AVP neurons are glutamatergic in primates. Both ultrastructure and immunoreactivity can therefore be sufficiently preserved in macaque brain tissues stored long-term, initially for light microscopy. Taken together, these results suggest that this methodology could be applied to the human post-mortem brain and be very useful in translational research.
Highlights
Studies using macaque monkeys as model non-human primates are essential because of their high applicability to human clinical research which often requires the testing of experimental therapies in primates
We first validated by Western blot analysis the specificity for the vesicular glutamate transporter 2 (VGLUT2) and neurophysin II (NPII) antibodies in Japanese macaque monkeys
The current study demonstrates that, in the macaque brain, interpretable ultrastructure and adequate ultrastructural immunoreactivity are both preserved in brain tissue even after long-term storage at −25 ◦ C for conventional light microscopy, provided that the fixed tissues are post-fixed with 0.1% glutaraldehyde after the long-term storage
Summary
Studies using macaque monkeys as model non-human primates are essential because of their high applicability to human clinical research which often requires the testing of experimental therapies in primates. Medical science is urged to make minimal use of animals such as primates, and basic studies on non-human primates appear to be permitted only if the work could not be carried out in any other species. Tissues from primates are often essential to achieve relevant results, there are considerable problems in their use. AVP is produced mainly by magnocellular neurosecretory neurons in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus [3]. The expression and release of AVP by magnocellular neurosecretory neurons in the SON and PVN are regulated by physiological conditions, including plasma osmotic pressure and blood pressure [7]
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