Anat Cell Biol 2021; 54(3): 340-349
Published online September 30, 2021
Copyright © Korean Association of ANATOMISTS.
1Laboratory of Neurosciences, School of Medicine, Universidad de Santander, Bucaramanga, Colombia, 2Institute of Neurosciences of Castilla y León (INCYL), Laboratory of Neuroanatomy of the Peptidergic Systems, University of Salamanca, Salamanca, 3Grupo GIR BMD (Bases Moleculares del Desarrollo), University of Salamanca, Salamanca, Spain
Correspondence to:Ewing Duque-Díaz
Laboratory of Neurosciences, School of Medicine, Universidad de Santander, Bucaramanga 680003, Colombia
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Using highly specific antisera, the neuroanatomical distribution of folic acid (FA) and retinoic acid (RA) has been studied for the first time in the children brainstem. Neither immunoreactive structures containing RA nor immunoreactive fibers containing FA were found. FA-immunoreactive perikarya (fusiform, small/medium in size, one short dendrite) were only found in the pons in three regions: central gray, reticular formation, and locus coeruleus. The number of cell bodies decreased with age. In the first case studied (2 years), a moderate density of cell bodies was observed in the central gray and reticular formation, whereas a low density was found in the locus coeruleus. In the second case (6 years), a low density of these perikarya was observed in the central gray, reticular formation, and locus coeruleus. In the third case (7 years), a low density of FA-immunoreactive cell bodies was found in the central gray and reticular formation, whereas in the locus coeruleus no immunoreactive cell bodies were observed. The distribution of FA in the central nervous system of humans and monkeys is different and, in addition, in these species the vitamin was located in different parts of the nerve cells. The restricted distribution of FA suggests that the vitamin is involved in specific physiological mechanisms.
Keywords: Folic acid, Humans, Immunohistochemistry, Retinoic acid, Vitamins
Vitamins are complex structures with a variety of functions and, in nature, show a wide distribution. Although the metabolic role played by vitamins (acting as coenzymes or cofactors) in many reactions is well known [1, 2], the knowledge on the neuroanatomical localization of these molecules in the mammalian brain is lesser. In recent years, due to the development of new highly specific antibodies against vitamins, the neuroanatomical distribution of these compounds in the mammalian central nervous system has become a promising line of research [3-10]. In this sense, the distribution of fibers and cell bodies containing folic acid (FA), retinoic acid (RA) (a metabolite of retinol or vitamin A), thiamine (vitamin B1), riboflavin (vitamin B2), nicotinamide (the amide of nicotinic acid or vitamin B3), pantothenic acid (vitamin B5), pyridoxal/pyridoxine (natural forms of vitamin B6) or vitamin C has been reported in the brain of rats, monkeys and humans. Most of the previous studies were performed in the central nervous system of monkeys [3-9, 11]. The knowledge on the anatomical distribution of vitamins leads to gain insight into the role played by these compounds (
FA acts as cofactor/coenzyme (
According to the data reported above, no information is currently available on the neuroanatomical distribution of FA and RA in the human brain. Thus, in order to increase our knowledge about this distribution our aim is to study by using immunohistochemical techniques the distribution of both vitamins in humans. The results of this study will serve to gain information about the possible unsuspected physiological actions mediated by both vitamins.
The experimental design, procedures and protocols of this work have been performed under the guidelines of the ethic and legal recommendations of the Colombian legislation (Resol. 8430/1993) and in accordance with the Helsinki declaration. Furthermore, this work was approved by the Ethic Committee of the University of Santander (UDES, Bucaramanga, Colombia) (Act 023-17). In this study, three brains of male children (2, 6, and 7 years old) who died respectively from respiratory failure, dengue, and drowning by immersion were used. According to the Colombian legislation brains were collected, from routine autopsies, within 24–48 hours after death (University of Santander, Bucaramanga, Colombia). In all cases, the pathological reports showed no evidence of neurological diseases.
The brain of each subject was removed and later the brainstem was dissected out. As previously described , brainstems were immersed in 10% formalin (three weeks at 4°C) to keep them preserved and then transferred to 4% formaldehyde in 0.1 M phosphate buffer-saline (PBS; pH 7.4) (for 30 days at 4ºC). After fixation, brainstems were kept in PBS at 4ºC and cryoprotected in increasing sucrose solutions (5%–30%) until they sank. Brainstems were cut using a cryostat (Leica CM1860): 50 µm thick coronal sections were taken, collected in PBS, kept at 4°C, and processed for immunostaining. One-in-six sections were mounted onto adhesive slides for Nissl staining.
As previously described [5, 6, 10, 27], free floating sections were treated with distilled water containing H2O2 (30%), NH3 (20%) and NaOH (1%) for 20 minutes in order to avoid interference with endogenous peroxidases . Sections were washed for 20 minutes in PBS (0.15 M; pH 7.2) and pre-incubated for 30 minutes in PBS containing Triton X-100 (0.3%) and normal horse serum (2%). Sections were incubated overnight (4°C) in the above mixture containing the anti-FA antiserum (diluted 1:500) or the anti-RA antiserum (diluted 1:500). Following this, sections were washed in PBS (30 minutes) and incubated at room temperature with biotinylated anti-rat (FA) or anti-rabbit (RA) immunogammaglobulins (Vector; Vector Laboratories, Burlingame, CA, USA) diluted 1/200 in PBS (60 minutes). Then, sections were washed in PBS (30 minutes), incubated for 60 minutes with avidin-biotin-peroxidase complex (Vectastain, Vectastain standard ABC kit, diluted 1/100; Vector Laboratories), washed in PBS (30 minutes) and Tris-HCl buffer (pH 7.6; 15 minutes) and the tissue-bound peroxidase was developed with H2O2 using 3, 3’ diaminobenzidine as chromogen. Finally, sections were rinsed with PBS, dehydrated and coverslipped with Entellan.
In addition, in brainstem sections the heat-induced epitope retrieval (HIER) method was performed . Thus, a container with Tris-EDTA recovery buffer (10 nM Tris, 1 mM EDTA, 0.05% Triton X-100, pH 9.0) was preheated in a rice cooker with water until reaching 97°C. Then, sections were carefully soaked in the buffer for 40 minutes. Later, the container with the samples was removed and the tap water was allowed to run for about 10 minutes until it cools. Finally, sections were removed and immersed in PBS until the immunohistochemistry technique was performed.
The immunological properties of the polyclonal primary FA and RA antisera used here have been reported previously in monkeys (FA)  and rats (RA) . Both antibodies were purchased from commercial sources (Gemacbio S.A., Saint Jean d’Illac, France) and were raised in rats (FA, reference AP099) and rabbits (RA, reference: AP057) with their respective bovine serum albumin (BSA) immunogens. Thus, rats/rabbits were immunized by one injection every 2–3 weeks. Each administration (subcutaneous injection) contained a mixture of 250 µl of complete (only used in the first immunization)/incomplete Freund adjuvant and 250 µl of an immunogenic NaCl solution [4, 8]. Serum samples were collected every three weeks and the antisera were pre-purified by immunoabsorption and precipitated. Later, the antibodies raised were characterized in ELISA tests [6, 10]. In both cases (FA and RA), the antibody avidity (IC50) was rather high (10–8 M).
As previously reported [6, 10] and in order to prevent non-specific immunoreactivity due to the anti-carrier antibodies, the first antiserum was preabsorbed (before the immunohistochemical application) with the respective coupling agent and carrier protein. Moreover, as previously reported , the specificity of the anti-FA antibody was very high, since the antiserum discriminated conjugated FA from other conjugated molecules (
According to previous works [5, 27, 30], the atlas of Haines (2012)  was used for mapping and nomenclature. One out of six sections was routinely stained for Nissl substance with cresyl violet to delineate the brainstem nuclei in which the immunoreactivity was observed. To determine the density of the immunoreactive cell bodies in the children brainstem, perikarya were graded into three categories as previously reported : high (more than 20 cell bodies/region/section), moderate (10–20 cell bodies /region/section) and low (less than 10 cell bodies/region/section). According to the size of the cell body and, as previously described , immunoreactive cell bodies were considered large (>25 µm in diameter), medium-size (15–25 µm) and small (<15 µm). Photomicrographs were obtained with an Olympus DP21 digital camera attached to an Olympus BX43 microscope (Olympus, Tokyo, Japan). To improve the visualization of the results, only the contrast and brightness of the images were adjusted (Adobe Photoshop CS6 Software; Adobe, San Jose, CA, USA), without any further manipulation of the photographs.
To determine the number and average of immunoreactive cell bodies, images were digitized according to the imageJ software (developed by the NIH) and available free of charge on internet . The colour images for each case (2, 6, and 7 years) were loaded into the imageJ software and a duplicate was made to work on it. Then, a scale adjustment was made according to the objective with which the images were captured to calibrate the section area. Following this, shadows were eliminated and the region to be treated was delimited. The photographs were converted into an 8-bit image type, followed by a threshold adjustment and binarization so that only the counting points remained. Finally, a particle analysis was carried out, giving the sum and average of perikarya.
Neither immunoreactive structures containing RA (fibers or cell bodies) nor FA-immunoreactive fibers were observed in the children brainstem. Thus, only cell bodies containing FA were found and they were exclusively located in the pons at the level showed in Fig. 1. It is important to remark that no difference was observed in the distribution of cell bodies containing FA in the children brainstem when the HIER method was applied or not. In addition, after applying this method, no immunoreactive structure containing RA was visualized. In all cases, cell bodies containing FA were fusiform, small/medium in size and showed in general one short dendrite. In addition, FA-immunoreactive cells bodies showed a restricted distribution: they were only observed in the central gray, locus coeruleus and reticular formation (above the dorsal trigeminothalamic tract). It is important to note that the number of the immunoreactive cell bodies varied depending on the age (Table 1). Thus, it was observed that as the age of the subjects increased, the number of the immunoreactive-cell bodies decreased (Figs. 1, 2A–C, 3); however, in the three cases studied the intensity of the immunoreactivity was similar.
In the first case (2 years), a moderate density of cell bodies was observed in the central gray (Figs. 1A, 2A, 3A, B) and in the reticular formation located above the dorsal trigeminothalamic tract (Fig. 2A), whereas a low density was found in the locus coeruleus (Figs. 2A, 4A). In the second case (6 years), a low density of these perikarya was observed in the central gray, above the dorsal trigeminothalamic tract (reticular formation) and in the locus coeruleus (Figs. 1B, 2B, 3C, D, 4B). In the third case (7 years), a low density of FA-immunoreactive cell bodies was found in the central gray (Figs. 1C, 2C, 3E, F) and reticular formation, whereas in the locus coeruleus no immunoreactive cell bodies were observed.
Table 1 shows the number and density of cell bodies containing FA in the human brainstem. Thus, the digital analysis of images obtained (eight for each age) showed that for the two-year-old subject, the total number of immunoreactive cell bodies in the central gray was 117 (mean 14.625), while for the reticular formation and locus coeruleus was 87 (mean 10.75) and 12 (mean 1.50), respectively. For the six-year-old subject, the total number of immunoreactive perikarya observed was 47 (mean 5.875) in the central gray, 34 (mean 4.25) in the reticular formation and 8 (mean 1.00) in the locus coeruleus. Finally, for the seven-year-old subject, a total number of 14 (mean 1.750) immunoreactive cell bodies were observed in the central gray, whereas in the reticular formation 1 (mean 0.125) immunoreactive cell body was observed. At this age, no immunolabelled cell bodies were observed in the locus coeruleus.
According to the neuroanatomical distribution of vitamins in the mammalian brain, it has been suggested that these compounds could play more important actions than their well-known metabolic functions [3-6, 10]. Thus, studies focused on the neuroanatomical distribution of vitamins in the mammalian central nervous system are important because, according to this distribution, unexpected actions of these compounds could be discovered. Here, the mapping of FA in the human brainstem is described for the first time. Immunohistochemistry is a suitable tool to increase the knowledge on the distribution of vitamins in the mammalian central nervous system and, in the future, the neuroanatomical findings reported here will contribute to know the physiological functions in which FA is involved in the human pons.
In the last decade, the mapping of immunoreactive structures containing vitamins (
To date, only one study has reported the neuroanatomical distribution of a vitamin in humans ; in this case, the distribution of vitamin C was performed in the brainstem of children during postnatal development . In the latter study two groups of ages (younger and older than one year of life) were compared. Like here, immunoreactivity for vitamin C was only found in cell bodies, but the distribution/number of these perikarya was more widespread in older children and this means that the ability to retain vitamin C is maintained or increased with age. However, the opposite effect was observed for FA: the number of neurons containing FA was higher in younger children. Although, due to the low number of cases studied here (one per age), the latter observation must be confirmed in future studies. Moreover, the distribution of cell bodies containing vitamin C was widespread in the brainstem of children, whereas in the same region of the central nervous system the distribution of FA-immunoreactive perikarya was very restricted. In fact, to date, in the children brainstem vitamin C showed the most widespread distribution of a vitamin in the mammalian central nervous system. It is important to note that cell bodies containing vitamin C were observed in the three pontine regions in which FA-immunoreactive perikarya were observed; thus, a colocalization of cell bodies containing FA or vitamin C occurs. According to the morphological characteristics of the immunoreactive perikarya (
The specificity of the immunoreactivity observed here, in addition to the histological controls performed, has previously been confirmed by using other techniques (
Currently, the functional and specific roles of FA in the children brainstem are unknown. Thus, more neuroanatomical and physiological studies are required to know the functional roles played by FA; however, the restricted distribution of the vitamin in the children brainstem suggests that FA is involved in specific physiological actions which may vary according to the nuclei/regions in which this vitamin was located. For example, the presence of FA in the locus coeruleus suggests that the vitamin could be involved in the modulation of catecholaminergic function (
In summary, the presence and distribution of cell bodies (fusiform, small/medium in size, showing one short dendrite) containing FA in the children brainstem have been described. It seems that the number of cell bodies decreases with age. This distribution was very restricted and immunoreactive perikarya were only observed in three regions of the pons. The distribution of FA in the central nervous system of humans and monkeys is different and, in addition, in these species FA was located in different parts of the nerve cells. In the children pons, the presence of FA suggests that the vitamin is involved in specific physiological mechanisms that must be elucidated in future studies.
This work has been supported by the Universidad de Santander UDES (PINLO/111702005-41823/EJ), Bucaramanga, Colombia and the “Programa XI: Programa de Financiación de Unidades de Excelencia de la Universidad de Salamanca”, Salamanca, Spain. The authors want to thank the Morphophysiology Department (School of Medicine, Universidad de Santander, UDES) for kindly providing the human samples used in this work and the Language Service (University of Salamanca, Salamanca, Spain) for supervising the English text. The authors wish to thank Dr. Edwin Gómez Ramírez (Laboratory of Embryology and Physiology, Militar University Nueva Granada, Campus Cajicá, Colombia) for technical assistance.
Conceptualization: EDD, RC. Data acquisition: EDD, RC. Data analysis or interpretation: EDD, RC. Drafting of the manuscript: EDD, RC. Critical revision of the manuscript: EDD, RC. Approval of the final version of the manuscript: all authors.
No potential conflict of interest relevant to this article was reported.