A primary cilium in oligodendrocytes: a fine structure signal of repairs in thalamic Osmotic Demyelination Syndrome (ODS)

ABSTRACT

A murine osmotic demyelination syndrome (ODS) model of the central nervous system included the relay thalamic ventral posterolateral (VPL) and ventral posteromedial (VPM) nuclei. Morphologic comparisons between treatments have revealed oligodendrocyte changes and, already 12 hours following the osmolality restoration, some heavily contrasted oligodendrocytes formed a unique intracellular primary cilium. This unique structure, found in vivo, in mature CNS oligodendrocytes, could account for a local awakening of some of the developmental proteome as it can be expressed in oligodendrocyte precursor cells. This resilience accompanied the emergence of arl13b protein expression along with restoration of nerve cell body axon hillocks shown in a previous issue of this journal. Additionally, the return of several thalamic oligodendrocyte fine features (nucleus, organelles) was shown 36 h later, including some mitosis. Those cell restorations and recognized translational activities comforted that local repairs could again take place, due to oligodendrocyte resilience after ODS instead or added to a postulated immigration of oligodendrocyte precursor cells distant from the sites of myelinolysis.

KEYWORDS:

• Murine – thalamus –osmotic demyelination syndrome – oligodendrocyte

Introduction

First described by Adams and collaborators,^1,2 the osmotic demyelination syndrome (ODS) in the central nervous system (CNS) is a non-inflammatory neuropathologic condition accompanied by a broad clinical symptomatology: slight confusion, disorientation, deafness, memory loss to seizure, paresis, unresponsiveness and eventually coma, depending on the degree of myelin loss in the pons as ‘central pontine myelinolysis’ (CPM) and ‘extrapontine myelin’ (EPM) lesions.^1–8 The ODS neuropathologic syndrome is caused by a hastily adjustment of a temporary or chronic deficiency of the homeostatic sodium gradient as it can be pertinent to diverse homeostatic hormonal sodic perturbations such as those afflicted by exaggerated body fluid losses and emergently treated for alcoholism, diabetes as well as to defective dialysis.^1–3,9–14 A large reference list of ODS literature was recently cited^15–19 and it appeared that EPM lesions can appear before any CPM ones^19–25 and ODS in human EPM cases would be more frequent than CPM ones.²³ There, ODS can frequently appear clinically in the elderly, hospitalized population.^3,9,26 The high incidence noted in the recent years is probably caused by a more frequent magnetic resonance imaging (MRI) utilization for neurologic diagnostics in miscellaneous conditions.^8,14,21–30

Found or induced in small mammals,^31,32 ODS research studies have been done with rats and dogs before mice.^4,31–41 Future development of genetically modified mouse models may further clarify the pathophysiology of ODS, its associated disorders, as well as its resolution. Several murine investigations have already provided clarifications on the ODS trigger events and, there, a breach in the blood-brain barrier (BBB) suggested a leak of some circulating compound(s)¹⁹ that eventually would trigger astrocytic signaling(s) toward oligodendrocytes causing a rapid myelin loss of APC (Adenomatous Polyposis Coli) and proteolipid protein (PLP) immunoreactivity that culminated 48 h post-correction of hyponatremia. Those changes were revealed along with astrogliosis as well as with clasmatodendrosis (cell body swelling with fragmentation of distal processes^16,17,42). They were linked to other myelin repression or damages of molecular markers and losses^15–17,43 accompanied by a local, restricted microglial cell’s reactivity,^15–19 verified by ultrastructure.^15–17 In ODS, the delimited CNS EPM cytotoxic events of the thalamic injuries do not involve irreversible damages in the CNS neurons, demonstrating resilience in the same demyelinated regions.¹⁸ Others have observed LIF (Leukemia Inhibitory Factor), secreted at ODS48h by both astrocytes and microglial cells,⁸ as stimulating myelination can suggest healing out of ODS. Moreover, clinical ODS cases can heal after a proper rebalancing osmolarity.^8,20 Consequently, one would consider that myelination could also undergo some restitution in the ODS-susceptible regions of the CNS after myelinolysis where loss of axon parts along with macroglial damages have been recognized.^15–18

This report again analyzes the same groups of mice that have revealed ODS extrapontine tissue’s changes in^15–18 and further comforts all those data of this murine model of osmotic demyelinating syndrome (ODS) where the relay ventral posterolateral (VPL) and ventral posteromedial (VPM) thalamic nuclei have been shown susceptible toward osmotic-induced demyelination.¹⁹ It adds information about ongoing ODS oligodendrocyte changes and repairs. At 12-hour post rebalancing osmolarity, ultrastructural examination revealed a primary or single cilium in oligodendrocytes in vivo while morphologic hyperchromaticity have been featured as in.^15–17 In addition to this appearance, the expression of the arl13b protein was tested to be coincident with ciliogenesis, internal trafficking, axon guidance built-ups.^44–50 This unique protein label was also seen in the neurons corroborating the axon hillocks regrowth [18 and in preparation]. A primary cilium of oligodendrocyte has only been remarkably found in vitro where growth factors or hormones have been involved, supporting oligodendrocyte precursor cell appearance.^51–53 Therefore, our ultrastructural observations along with the exceptional finding of a primary cilium in vivo tend to support the hypothesis that this transient primary cilium would be one of the indicators for these macroglial cells to have undergone or that are undergoing some local in vivo ‘rejuvenation’ followed by differentiation events after ODS before and along myelin repairs that resume and carry on later.

Materials and Methods

The animals and the murine ODS protocol

Male C57bl/6 J mice, aged from 3 to 4 months, kept in the University Animal Facility, according to the experimental ODS protocol conducted in compliance with the European Communities Council Directives for Animal Experiment (2010/63/EU, 86/609/EEC and 87–848/EEC), approved by the Animal Ethics Committee of University of Namur (ethic project n814–210).

ODS induction was based on the correction of a chronic hyponatremia, adapted protocol from,³⁶ as described in.^15–18 Briefly, an osmotic minipump (Model 1004, Alzet, Cupertino, CA) was filled with desmopressin acetate (2 µg/ml; Minirin, Ferring, Saint-Prex, Switzerland) and inserted subcutaneously under anesthesia into the back of animals at day 0. Standard pellets and water were switched to a low-sodium liquid diet (AIN76A, MP Biomedicals, Santa Ana, CA), given ad libitum for the whole duration of hyponatremia. At day 4, hyponatremia level and serum sodium were returned to normonatremia using a single intraperitoneal injection of NaCl 1 M (1.5 ml/100 g body weight). Minipumps were left in animals until the end of experiments, as sacrificed, involving anesthesia performed using an intraperitoneal injection of a cocktail of ketamine 100 mg/kg and xylazine 5 mg/kg.

Experiment groups

This fine structure investigation complements others made with neurophysiology, histology, immunohistochemistry and molecular biochemistry^15–19 included four groups. Group 1 were normonatremic or Sham mice (NN; n = 13) sacrificed at day 0; Group 2 were hyponatremic mice (HN; n = 11) sacrificed 4 days after the induction of hyponatremia (day 0 + 4-day treatment period named ‘chronic hyponatremia’ as described in the ODS protocol (in a) and in.^15–19) Groups 3 and Group 4 were mice that underwent the 4-day ‘chronic hyponatremia’ abruptly provided with normonatremia as both ODS groups. Group 3 included mice sacrificed 12 h after a fast restoration of normal natremia, thus named ODS 12 h group (ODS12h; n = 6) while Group 4 mice encompassed mice sacrificed 48 h post fast osmotic correction, hence named ODS 48 h (n = 9). Under anaesthesia, all the mice were exsanguinated and perfused transcardially with warm NaCl 0.9% followed by phosphate-buffered 4% paraformaldehyde (PFA). Brains were removed, divided into two hemispheres and post-fixed overnight in the same PFA fixative solution. Out of NN, HN and ODS12h groups, studied in,^15–19 two half mouse brains were taken at random from each Group 1 to 3 and, from Group 4, three out of nine half brains were taken for ultrastructure analyses (as described in c.2, similarly to those studied in^15–19) (Fig. 1 and Table 1).

Figure 1. Experiments performed on 4 groups of 2 mice excepted for ODS 48 h which included 3 mice.Normonatremic mice (NN) from group 1 were sacrificed at day 0 (arrow) while uncorrected hyponatremicmice (HN) were sacrificed 4 days after the induction of hyponatremia (arrow). ODS mice were sacrificed asgroups 3 and 4, at respectively 12 and 48 h post correction, (arrows) similar to [15, 16, 18] studies

Table 1. Brain’s Morphology Analyses in the Mice Groups of this study, based on^15−18.

Mice Groups	nb	LM arl13b label of oligodendrocyte primary cilium	nb TEM	Primary cilium TEM
Normonatremic (NN)	13	-	2	-
Hyponatremic (HN)	6	-	2	-
ODS12h	11	+++	2	+++
ODS48h	9	-	3	-

nb: number of brains and analysed parts of same thalamic regions

Microscopic anatomy

C.1. Light microscopy (LM)

For histology, all the half brains were then dehydrated, paraffin-embedded and sectioned into 6-µm thick microscopic preparations stained with hemalum and chromoxane cyanine R or Eriochrome C for general topographic observation of nuclei and myelin.^15–18,54 Adjacent sections were used in other immunochemistry studies.

C.2. LM Immunohistochemistry

Semi-serial paraffin sections were dewaxed, rehydrated and heat-induced antigen retrieval was performed in citrate buffer pH 6 at 100°C for 10 min. Endogenous peroxidase was quenched using 3% H₂O₂ in methanol for 10 min. Nonspecific binding was blocked using 5% horse or goat serum diluted in Tris-buffered saline (TBS) for 15 min. Microscopic preparations were incubated overnight at 4°C with primary antibodies for either the oligodendrocyte marker p25α (1:1000; Sigma HPA036576) and for the marker of ciliogenesis arl13b (1:1365; Proteintech, Fisher Sc. 11711-1-AP), both diluted in TBS containing 1% normal serum. The next day, sections were incubated with a biotinylated secondary antibody (1:100; Vectastain, Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature.⁴⁴ Then, sections were incubated with a solution of peroxidase-bound streptavidin (1:100; Vectastain) for 45 min. Immunoreactivity was revealed using diaminobenzidine (Dako, Glostrup, Denmark) and hemalum was used as counterstain. Finally, dehydrated and mounted in DPX sections were observed with an Olympus BX63 microscope Olympus, Tokyo, Japan) equipped with Olympus SC50 camera. Images were acquired with the Cell Sens Software.

Transmission Electron microscopy (TEM)

The other half brains, already perfused transcardially, were fixed with a solution of PFA 2% and glutaraldehyde 2% in 0.1 M phosphate buffer (pH 7.4). The samples, already examined in previous reports,^15–18 were the thalamus ventral posterolateral (VPL) and ventral posteromedial (VPM) regions that have been harvested using a neurological punch of 0.69 mm of internal diameter (Fine Science Tools #18036-19, Heidelberg, Germany) along lateral plans 1.0 to 2.0 mm from interhemispheric fissure, according to the mouse brain atlas of Franklin and Paxinos.⁵⁵ The excised samples were then post-fixed in glutaraldehyde 4% for 2 hr, washed in Millonig’s buffer containing 0.5% sucrose for 24 hr, post-fixed in OsO4 2%, dehydrated and finally embedded in epoxy resin. Semi-thin sections, stained with toluidine blue, allowed to select regions of fine structure analysis. Ultrathin gray sections (ranging from 40 to 70 nm) of these regions, obtained with a diamond knife, collected on 200 and 300 mesh nickel grids (Micro to Nano, Haarlem, Netherlands) and contrasted with uranyl acetate and lead citrate were observed with a Philips Tecnai 10 electron microscope, at an accelerating voltage of 60–80 kV, equipped with a digitized Olympus ITEM platform MegaView G2 image analysis.

Results

Microscopic Anatomy aspects

1.a. LM of paraffin sections

Figure 2 is a set of microscopic anatomy aspects of mice NN, HN, ODS 12 h and ODS 48 h brain sections, stained with hematoxylin and Eriochrome C. The damaged thalamus region in ODS48h treated mice is evidently revealed by its poor sustainability compared with those of NN, HN and ODS12h thalami where no obvious difference in myelin staining was observed with LM and no clear-cut delineation was found. After chronic hyponatremia and its rapid readjustment to normonatremia. the ODS 48 h brain sections were, at that stage, those that revealed a large thalamus zone that had undergone myelinolysis as LM histopathological changes that can be identified.

Figure 2. Sagittal mice brain’s paraffin sections of normonatremic (NN), hyponatremic (HN), 12h aftercorrection of hyponatremia (ODS12h) and 48h after correction of hyponatremia (ODS48h). All stained withhemalum and eriochrome cyanine where the poorly stained ODS 48h section best revealed the thalamus (Th) overall myelinolysis

nb: number of brains and analyzed parts of same thalamic regions

1.b. LM of semi-thin epoxy sections

In both the ventral posterolateral (VPL) and ventral posteromedial (VPM) thalamic regions

(the ventral posterior nucleus) 1-µm semi-thin sections, histology enabled us to recognize the different cell types within those regions investigated. Examples of each treatment-sampled regions are displayed in Figure 3 as Control/Sham treated or Normonatremic (NN), Hyponatremic (HN), 12-h post ODS or ODS12h, and 48-h ODS or ODS48h treated. There, neuron somata, macroglial (astrocytes and oligodendrocytes) and some spaces, have been exemplified and labeled. including capillaries and some of the hollows caused by myelinolysis neuropil. Following those treatments, neuron cell bodies, astrocytes, and oligodendrocytes as well as microgliocytes did not appear to show very obvious damage under light microscope examination save immunolabels, and then ultrastructure observations complemented the changes revealed by those molecular markers. However, after comparisons and scrutiny of the oligodendrocytes found in all the semi-thin sections, the HN treated suggested changes in overall cell morphology and topology of the NN nucleus aspects, as illustrated in Figure 3 HN. The same cells recognized in ODS12h treatment displayed the highest basophilic nucleus contrast while those of ODS48h are with euchromatic aspects, more like the NN type, whether they were satellites or interfibrillar oligodendrocytes (Fig 3 ODS48h).

Figure 3. Comparison pane between 1 µm semi-thin sections stained by toluidine blue of representativeNormonatremic, Hyponatremic, ODS 12h, and ODS 48 h murine thalamus where some examples ofastrocytes (A), neurons (N), oligodendrocytes (O) and blood vessels (V) lined by endothelium (E) aremarked. Some empty spaces, marked by stars, are examples of some of the parenchymal cavities resultingfrom myelinolysis. Scales equals to 20 µm for all micrographs

1.c. LM p25α Immunohistochemistry

The patterns of marking the p25α immunolabel for mature oligodendrocytes reflected the toluidine blue sections and those of the ultrastructure findings: the detection of high number of cells occurred in NN and HN treatments while the labeled cells decreased but a few after ODS. Very few strongly marked cells were noted throughout the ventral posterior thalamus regions in ODS12h, likewise in ODS48h time lapse after rebalancing osmolality but with a large number of tiny, scattered staining regions (Fig 4).

Figure 4. LM p25 alpha immunolabeled patterns of oligodendrocytes of the NN, HN, ODS12h and ODS48hmurine thalamus. The dark-brown stained oligodendrocytes contrasted from other cells, including theadjacent large neuron cell bodies and decreased in fields examined in a sequence NN = HN > ODS48h >ODS12h. In the ODS48h, some labeled parts of oligodendrocytes appeared throughout the large lowerright, adjacent myelinolytic zone appearing as a poorly contrasted quadrant among mainly neuron cellbodies, save the outskirts, where one large cell is labelled. Scale in ODS48h for all sections equals 50 µm

Ultrastructural aspects

The observations have been taken out of collections of oligodendrocytes obtained from each of the treatments, out of zones selected from 1 µm-thick sections samples.

2.a. Control/Sham or Normonatremic (NN) oligodendrocytes

The oligodendrocyte cells typically revealed a pear- to round-shaped and revealed a more electron dense contrasted cytoplasm than any other cell seen with ultrastructure in the CNS (Fig 5A-C). The euchromatic, spherical nucleus is often seen in an eccentric position in a pear-shaped perikaryon where innumerable free or attached polyribosomes to short cisterns of endoplasm are speckled among other organelles such as short, ellipsoid mitochondria, a Golgi apparatus and its processing saccules distributed around the nucleus envelope in the remaining narrow perikaryon with sections of microtubules. Moreover, according to some lucky section views, either an entire centrosome, or one of both centrioles, involved in the transfer of outward products by microtubules, dispatched for the maintenance of the many myelin sheaths wrapping the axonal extensions in the adjacent neuropil Fig 5(A-C). An example of oligodendrocyte satellite showed with typical formed junctions with astrocytes and neuron cell bodies as in Figure 5A-B while interfibrillar oligodendrocyte associated with its adjacent maintained axons and can form junctional complexes with adjacent astrocyte (Fig 5C).

Figure 5. Normonatremic (NN) murine thalamus. A-B: Satellite oligodendrocyte (O) exposed its joined association with a neuron (N) and an astrocyte (As) forming a functional trio; in C: interfibrillar one. A:Astrocyte adjacent to a capillary (C) basal lamina surrounded by myelinated axons and other structures. B:Enlarged view of A; oligodendrocyte where a closeup of its gap junction with the astrocyte is evidenced (white arrow). Insert shows details of perikaryon with ribosomes. C: Interfibrillar oligodendrocyte crownedby some of its maintained myelinated axons and other adjacent structures of the neuropil

2.b. Hyponatremic (HN) oligodendrocytes

These cells appeared with some changed topology. The cytoplasm electron density is preserved as in the NN cells. In the example illustrated in Figure 6A,B, the nucleus distorted profile ultrastructure was somewhat elongated but revealed a compact, segregated nucleolus. Indeed, its components, i.e. the dense fibrillar component compacted as a thick band is prominently distinguished and tend to separate from the granular accumulated cloud or spread of ribonucleoproteins that reached the peripheral layers of heterochromatin, decorating the inner nuclear leaflet of the nucleus envelope. In the example taken in Fig 6B, an adjacent, contacted neuron cell body maintained a large Golgi apparatus while the adjacent surrounding neuropil displayed some swollen myelinated axons and other neurite’s extensions. The electron-dense, shrunk oligodendrocytes displayed junctional contacts with astrocytes along with, in many samples examined, peculiar tiny or small intercellular parenchymal rifts (Fig. 6A-B).

Figure 6. Hyponatremic [HN] satellite oligodendrocytes. Either electron dense cytoplasm with a showed topologic change of the oligodendrocyte nu cleus in A. and B as a lesser size section. Oligodendrocytes maintain junctional complex (black arrows) with adjacent neurons (N), recognized with large Golgi. Axonal myelinated profiles of diverse orientations are associated with oligodendrocytes (white arrows) in A; As: Astrocyte edge. Numerous small and enlarged intercellular neuropil spaces are found near each oligodendrocyte extensions in B (open white arrows in B)

Figure 7. TEM aspects of ODS12h oligodendrocytes (O) from a demyelinating area of the mouse thalamus showing an enhanced cell contrast of both nucleus and cytoplasm. Displays of rare, scattered cisterns of endoplasm (in A-C) or clustered in the perikaryon and in parts of the extensions (also see in 7DF). The enlarged mitochondria profiles (white arrows in 7B) are reaching associated axons where myelin revealed adjacent neuropile blemishes, including discrete intercellular parenchymal spaces (black arrows in B and C). Associated with an astrocyte (As) in A, an insert of a small area (dotted square) is enlarged to illustrate the astrocyte-oligodendrocyte junctional membranes. B and C: Aspects of condensed heterochromatin where, among the contrasted, mottled nucleoplasm interchromatin more intensely contrasted granules appear. Note in all adjacent associated still myelinated axons

Figure 8. LM and TEM ODS12h oligodendrocytes. Detected at first with TEM in its narrow pocket (white arrow in B), it was later revealed with the arl13b immunolabel, and further illustrated in TEM Figures 9 to 12C. A: LM paraffin section of the thalamus region of B that revealed the arl13b immunolabeled proteins, constituting two primary cilium profiles; scale equals 2µm. B: TEM aspect of an ‘hidden’ primary cilium (underlined white arrow). Open white arrows mark astrocyte-oligodendrocyte junctional contact zones (plakins and associated macromolecules) while black arrows indicate some of the delicate oligodendrocyte extensions with enlarged mitochondria throughout the surrounding neuropil, linking them with their axon’s myelin. As: Astrocyte; O: Oligodendrocyte

2.c. ODS 12 h oligodendrocytes and arl13b immunolabel

Half-day after the fast correction of hyponatremia, oligodendrocytes were easily recognized throughout the samples examined because of their evident increased chromaticity compared with both NN and HN cells. There, nuclei, and the surrounding cytoplasm, were still heavily electron-contrasted. The compaction of heterochromatin is progressive enough to occupy a large part of the nucleoplasm while other parts were crammed with a mottled darkish and grainy appearance (Fig 7A-F). There, loose but still contrasted nucleolar masses could only be recognized as small aggregates among haphazardly dispersed interchromatin granules or masses in the grayish, lesser contrasted nucleoplasm (Figs 7A-F). Peculiarly, these interchromatin zones revealed several alignments of particulate compounds that have nothing to do with sectioning artifacts (arrowheads in Fig 7F). Narrow paler-contrasted regions, among the most contrasted heterochromatin, revealed the nuclear pore regions as typical electron-lucent spaces. In this group of ODS12h thalami, not LM but only electron microscopy was able to make us detect in the demyelinated zone. With fine structure survey, all these ODS12h cells depicted a progressively contrasted cytoplasm with grouping of small round vacuoles and a few autophagocytosis features while a centriole or both of the centrosome can become detectable (i.e. Fig 7A-F). In all ODS12h oligodendrocytes, enlarged mitochondria became more obvious in the perikaryon were also recognized in the outermost cell parts, including in the narrowest extensions reaching throughout and among the adjacent neuropil the associated myelinated axons (Fig. 7B-C and insert).

Figure 9. ODS12h oligodendrocyte aspect of Figure 8 primary cilium. A: Axoneme where the tip microtubule ends (arrow) appears ‘cut’ by the endocytic formed structure due to the sectioning angle. The open arrow indicates the narrow, demarcated cilium pocket formed by fusing coated vesicles near its shaft region. Note the contrasted cytoplasm with scattered ribosomes and mitochondria profiles. As: Astrocyte; Ax: Axon and its myelin

Remarkably, in some of these densely contrasted oligodendrocyte profiles, random sections made us able to detect an internal or enclosed ‘primary cilium’ structure, revealed in Figure 8A-11) among the already contrasted cytoplasm. The detection at the low TEM magnification was unclear, due to the heavy contrast background cytoplasm and because this peculiar cell appendage and other associated structures had only a narrow gap envelope and furrow. It was only with an enlarged or high magnification that the recognition of an almost entire primary cilium view in those oligodendrocytes was possible. The serially sectioned parts had grid bars impeding the following section plane views. However, other parts of cells, similarly contrasted displayed parts of primary cilium, were exclusively displayed in the ODS12h group and not in the others. Out of 20 ODS12h oligodendrocytes, five aspects of primary cilium, however different and partial cell features were collected but not depicted entirely, save in LM, but with lack of details and displayed in cross- and almost entirely longitudinal aspects (Fig 8A). Meanwhile, enough of the fine resolution recorded allowed us to recognize morphologically the primary cilium (Figs 8B-11) as other aspects seen in other micrographs would allow us to note the accompanying and associated organelles (Figs 10-12C). Enlarged, inflated-like mitochondria profiles with fine cristae depicted the many mitochondria profiles that could reach at least twice the size of the NN or HN ones. Scrutinizing these micrographic fields of view, perikaryal zones can be viewed with extended branching but delicate extensions in the neuropil and contained at least one large lipofuscin body and featured one microtubule core bundle shaft and the basal, densely contrasted root patch zones were noted had probably organized out of one of the centriole bodies of the centrosome, deeply located in the perikaryon (Figs 7A, 10-12C). This root as apparent electron dense but specialized structure that serve as a footing for the assembled microtubules and showed elongation as in its axonemal structure. Finally, a sort of internal appendage ‘primary cilium’ was recognized, even though the central microtubules were not verified in cross-sections (Figs 11-12A). However, a surrounded space widened because small, round-shaped vesicles either alone or aggregated into an irregular void with heterogeneous internal contrast and heterogeneous membrane density was seen (Figs. 10-12A-C). There, a fuzzy internal coat fused with the created internal space. It is noteworthy to point those vacuoles, whose content appeared electron lucent but by places, they revealed a heterogeneous membrane: rim parts were heavily contrasted with or without internal fuzzy and highly contrast, suggestive of peculiar glycoprotein or glycolipid content (Figs 7-12). They have been called ‘ intraflagellar transport vesicles’ (IFT) in other published studies, even though there is no ‘flagellum’ but a peculiar cilium. Among these organelles, segments of Golgi apparatus and smooth endoplasmic reticulum were not well distinguished while small segments of the rough endoplasmic reticulum bore a coat of a few ribosomes, hanging by their mRNA treads or linkers (Figures 9, 10 and 12C). The lipofuscin body/lysosome adjacent could result from membrane lipid turnover as some vesicles could be the result of autophagosomes because the IFT vesicles are associated with this primary cilium building or maintenance. Hence, all these fine structures found in the random ultrathin sections allowed us to identify a primary cilium in a mature thalamus oligodendrocyte.

Figure 10. ODS12h oligodendrocyte primary cilium: Basal body (BB) zone and cilium pocket basal edge (open arrow); it is shown with an irregularly shaped fusing vesicle issued from fused aggregated small ones (white arrow), surrounding its axoneme. Black arrows indicate two evident polyribosomes

The same thalamus region that appeared with LM myelinolysis is not evidently damaged by myelin in ODS12h but regional axon profiles displayed unwrapped or scratched myelin lamellae segments, dilated and with enlarged, stretched mitochondria, swollen endoplasmic reticulum saccules and cytoskeletal poorly defined (neurotubules, neurofilaments). There are lysosomal bodies even though some axo-dendritic connections could be remained in the adjoining neuropil (Figs 7F and 11). Among the adjacent structures detected, some delicate parts of establishment of translational astrocytes were recognized by rare thick beta glycogen granules and maintained junctional contact zones, densely contrasted as shown in Figures 7A and especially in Figure 11C.

Figure 11. ODS12h oligodendrocyte sectioned profile away from the nucleus region where a tight microtubule fascicle (open arrow) is located among the contrasted cytoplasm. Organelles, including enlarged mitochondria (black arrows), lipofuscin body (Li), scattered ribosomes among spherical vesicles. Ax: Presynaptic axon region surrounded by some of the oligodendrocyte extensions (arrowheads). Stars indicated damaged axons in neuropil

ODS 48 h oligodendrocytes

Interfibrillar and satellite oligodendrocytes were both recognized (Figs 13A-C to 17A-B) along with rare mitosis (Fig 16A-B) and fine structure with the resulting twin cells (Fig 16C), resulting from replication with morphology of somewhat poorly differentiated cells while others, most often revealed an overall morphology almost similar to NN cells with a fine structure that showed a lower electron contrast compared to all NN, HN, and ODS 12 h. No primary cilium has been detected anymore at this stage of the experiment by electron microscopy but only an obvious centrosome or one centriole of it (Fig 13A and C). Possibly, after ‘survival’ both cell’s nucleus and cytoplasm appeared as less mature, differentiated and active, revitalized as both types of oligodendrocyte of the thalamic parenchyma seemed to have restored like the adjacent neurons (Figs 13A-C-14A-D, 15, 16B), through transcriptional and translational activities because their fine structure demonstrated a round to oval shape euchromatic subcentral nucleus, containing some peripheral heterochromatin and one or more NORs in the nucleoplasm, thus appeared very uniquely changed from the hyperchromatic, mottled-contrasted ODS 12 h oligodendrocytes. The perikaryon was also filled with innumerable dispersed translating ribonucleoproteins with a few large beta glycogen granules that surround abundant organelles (Figs 14A, 15, 17A-B). In Figure 14A, a nuclear envelope region, near its adjacent nuclear pore, appeared evaginated by a long, winding appendage making it a growth of a rough endoplasmic cistern plunging in the now ‘busy’ perikaryon. It is branching into fine parts, revealed by bit and segments of it where many ribosomes decorated evident small to elongated endoplasm parts and, thus, supportive of reestablished secretory activities that shall be important to later, repair the dependent myelin structures associated (Fig. 14 B-C). The perikaryon endoplasm also contained innumerable tiny coated vesicles and a large, dispatching endosome (Fig 17A-B). The Golgi apparatus remained difficult to discriminate from all the membranous vesicles, some acquired bizarre shapes. A few, rare tiny lysosome-like as wrapped bodies locate to the cell outskirts. It is the same for most mitochondria whose large size noted in ODS12h are few and seemed to have regained the morphology as those seen in NN cells. Among those mitochondria, some enlarged ones enclosed still swollen or blemished matrices and cleft parts, reminding the investigator of their past osmotic ‘scar’ or compensatory glycolytic adaptation as ODS defects and, perhaps, now depicting a remodeled aspect (Figs 17A-B). Furthermore, the obvious finding of centriolar bodies (centrosome) near the endosome seemed to comfort the reactivation of the contrasted, latent ODS12h oligodendrocytes for future microtubule cell networks. The current aspects seen in Figure 13C only short segments of microtubules, haphazardly distributed among the RER were noted among the organelles and innumerable poly- and ribosomes. Interestingly, many single or bundled axons in the neighboring oligodendrocytes or adjacent to them appeared myelinated or enclosed by the oligodendrocyte cytoplasm (Figs 13-17A-B) while in the same or other areas of the demyelinated zone, axon profiles and myelin still revealed blemishes and contained swollen and altered organelles. Other views from ODS48h fascicular oligodendrocytes in the area adjacent to the worst epicenter demyelinated zone showed cells with quasi typical NN morphology (Fig 13A, 14, 15, 17A). They contained large round euchromatic with LM found oligodendrocytes with euchromatic nucleus that included a typical nucleolus as for immature cells growth and the associated perikaryon aspects that encompassed abundant organelles, including a Golgi apparatus (Fig 17A-B). Furthermore, the same ODS48h oligodendrocyte cell bodies were able to display tiny astrocyte-oligodendrocyte junctional complex rim segments (arrow in Fig 17A). Additionally, several adjacent neurites were wrapped by oligodendrocyte extensions (Figs, 14A-D, 16B, 17A). In the parenchyma, oligodendrocyte satellites found similar perikaryon to swollen parts of mitochondria, caused by previous osmolyte disturbance. Intercalated between oligodendrocyte and neuron cell body, astrocyte extensions also contained some ODS defects, remains of astrogliosis

Figure 12. Detail views of Figure 10 illustrating the microtubule array (t) and the accumulation of small round, clear vesicles, some fusing with one another and adjacent within the microtubule alignment (arrows); some vesicles contained tiny segments with high contrast (white arrows) can also be viewed in B. Black arrows show autophagosomes resulting in lipofuscin body (li); mi: mitochondrial profile. The accumulated granules in the cytoplasm (as in top of A) are also scattered among the vesicles in B and C; they are polysomes, identified by their mRNA linkages, marked in C (slanted white arrows)

Oligodendrocyte replication in ODS 48 h thalamus

Among the one-µm thick sections, rare but disseminated mitotic oligodendrocytes were detected in ODS48h samples (e.g. Fig 16A). Those rare replication events, revealed in the semi-thin sections of the thalamic ODS regions investigated produced undifferentiated-like, ‘twin’ oligodendrocytes in the same regions of the thalamic CNS. Simultaneously. adjacent and functionally associated neurites can be noted enclosed by a few myelin layers as illustrated by these differentiated oligodendrocytes (Fig 16B).

Figure 13. Views of fine structures in one interfibrillar ODS 48h oligodendrocyte surrounded, in A, by bundles of axons where myelin juxtanodal zones still showed damages (small white arrows) and axons profiles with swollen content (star). In A and B: mitochondria still reveal swollen parts (black arrows). C: Enlarged perikaryon region decorated by innumerable ribosomal particles containing numerous organelles: a large endosome (E) and two obvious centriolar bodies are surrounded by numerous small coated vesicles and numerous haphazardly distributed, bended microtubule segments (white arrows). A black curved arrow marks a long winding RER cistern as an extension of the nuclear envelope

Discussion

4. a. The oligodendrocytes: development and myelin

Oligodendrocytes were identified by Del Rio-Hortega⁵⁶ and, afterward, numerous studies and reviews encompassed the growth, structure and functions of these CNS macroglial cells. Specialized interactions with the nerve cells and axons, the blood-brain barrier components, astrocytes as well as microglial cells have been abundantly described and reviewed.^57–75 From the earliest studies onwards, the demonstration of the CNS myelin formation and maintenance by the envelopment of nerve extensions by the oligodendrocytes was found more complex than those of the Schwann cells making myelin in the PNS because one oligodendrocyte can wrap many axon’s internodes instead of one Schwann cell wraps one internode structure in the PNS.^58,59 Additionally, the oligodendrocyte integrity influences diameter, insulating function for proper conduction along the associated nodes of Ranvier and the other adjacent substructures (paranode and juxta-node regions.^{59,60,62–73} Some of the dynamic interactive cell mechanisms of regulation found in vitro for differentiation of oligodendrocytes were comforted by in vivo in parts of the CNS.^{61,65–68,73–85} It is during embryonal development that oligodendrocytes undergo growth and differentiation out of at least two waves of precursors migrating out of a NG2 glial spinal cord cell type; one of them gives rise to motor neuron precursors before those of oligodendrocyte precursors developing into diverse pools of oligodendrocytes.^{60,62,65,66,68,73–84} Then, these precursor cells migrate throughout the CNS parenchyma regulated by series of growth and modulating factors.^78–85 Specifically, the murine wave of differentiation starts in the spinal cord at birth and follows a caudo-rostral direction toward the brain, with the neocortex as last region to be myelinated while in humans, myelination begins in the spinal cord at mid gestation and continues for at least the first two decades of life.⁸⁴ The development and differentiation steps of these cells have been deciphered until maturity through series of signaling factors stimuli regulating series of proteomic expressions that were clarified with in vitro experiments and in some recent, in vivo, knock out murine models.^69–71 Some of the differentiation processes can still be found in the postnatal, mature murine CNS^{69–71,82,83} and would involve some epigenetic modifications whose illustrative morphology is incomplete, especially with electron microscopy, in view of some of the recent modeling schemes that have been hypothesized.^84,85

Figure 14. ODS 48h oligodendrocyte satellites (O) and nerve cell bodies (N) of murine thalamus. A: Neuron with huge branched mitochondrial profile in a dendritic extension (long black arrow) surrounded by its oblique to cross-sectioned axonal profile extensions (white arrows). The dark, contrasted satellite oligodendrocyte has an extension that is seen wrapping a neurite profile at the low right quadrant (curved arrow), enlarged in B and C with the oligodendrocyte part filled by a transparent blue hue. A similar aspect of A, found in semi-thin sections is also depicted in D. V: capillary lumen

4.b. Oligodendrocytes and hyponatremia CNS fine features

The immunolabeling of oligodendrocytes with p25α, associated with the differentiated microtubule components of oligodendrocytes was as strong and consistent with typical content and the other LM and fine morphology in both NN and HN cells.^15,16,86,87 Comparing fine structure of both NN and HN cells, chronic hyponatremia did not impact on the cell’s electron density by processing the tissues. However, the electron contrast usually observed in the oligodendrocyte’s ultrastructure associates with cytoplasmic contents but is seldom explained either in most basic sciences or pathology textbooks. Oligodendrocytes can be usually identified by their small size and poor cytoplasm but with high electron density compared with all the other cells of the CNS, save microglial cells, also often loaded with many lysosomes.^58,88–92 Studies with noted oligodendrocyte electron contrast were deduced to be caused by three stages of maturation, from the least matured (lowest contrast) to the more mature or adult type (highest electron contrast).⁹³ Such ultrastructure findings were explained by Peters and others⁸⁸ as they have illustrated the oligodendrocytes to contain, as quoted: ‘a matrix of fine dense particles that are responsible for the inherent density of the oligodendrocytic cytoplasm’. Others identified these particles as iron in the form of heavy or H-ferritin that can be taken up by Tim-2 receptors^94,95 and iron uptake through transferrin by the normal BBB.^96,97 In ODS, the blood-brain barrier has been shown to be breached¹⁹ and, excessive, uncontrolled extracellular iron level could have induced an oxidative stress⁹⁸ that may have triggered changes in nucleus and cytoplasm of key cells of the brain parenchyma. The electron density of the oligodendrocyte nucleus may have resulted from those changes like in those found in experimental sodium depletion perturbing internal nucleus-cytoskeleton modification⁹⁸ and the channeling of iron⁹³ together with the dehydration processing of the samples. Both and other unclear factors could have made revealing a few enlarged neuropil intercellular spaces along with some possible astrocyte profile parts swollen. At a low and with control by the intact BBB, iron is involved in both functional astrocytes, oligodendrocytes and myelination as well as remyelination.⁹⁵

Figure 15. ODS48h oligodendrocyte satellite (O) of the murine thalamus with semi-thin insert of similar cell. Swelling damage remnants out of ODS are shown (white arrows to mitochondria) with abundant perinuclear ribosomes along with junctions with an intrusive astrocyte (As; long black open arrow) still with swollen parts. An adjacent neuron (N) shows lysosome bodies (i.e. ly) and exhibits intercellular junctions (open black arrows) with the oligodendrocyte. Among the parenchyma or neuropil, axon (Ax) and dendrites (d) are noted

4.c. 12-hour ODS oligodendrocytes

Twelve-hour after the fast [Na+] rebalancing, LM views of both oligodendrocyte semi-thin sectioned (stained by toluidine blue) and paraffin sections (labeled by p25alpha immunomarker) showed among the ODS thalamus scattered oligodendrocytes with strong basophilia and fully labeled cells for a suggestive peculiarly organized microtubular arrangement. These changes were verified in^15,16 along with astrogliosis, illustrated in¹⁷ as well as in this report by the unique discovery of a primary cilium ultrastructure.

Figure 16. Interfibrillar oligodendrocytes (O) of ODS48h murine thalamus. A-B: examples of mitotic cells (open arrows); A: in myelinolytic zone and B: in periphery of myelinolysis epicenter. Scales equal 5µm. C: TEM aspect of twin oligodendrocytes, resulting from mitosis amongst the repairing neuropil. Poor myelination wrappings and some blemishes from myelinolysis are marked by white arrows. As: Astrocyte, v: blood vessel; * : examples of wide neuropil spaces caused by myelinolysis

4.c1. Electron density

Among the causes of chromatin condensation of oligodendrocytes, one could presume that, in this in vivo experimental situation, a sort of ‘pyknosis’ transient status occurred as one described in cell basic histopathology because of the high-contrasted basophilia of the small cytoplasm and that of the nucleus hyperchromaticity that was built up. Meanwhile, the perikaryon had only small and few lysosomal bodies as lipofuscin as noted before and no autolysis nor deteriorating endoplasm aspects^15–17 but several small round, lucent vesicles stood out against the electron dense cytoplasm occupied by scattered ribosomes, some of them associated with small to enlarged curved clefts, making it wide or long rift. In this condition, the oligodendrocyte’s nucleus, following the chronic hyponatremia status depletion endured a dehydration step³⁹ and, after a fast replenishment of [Na+] in the CNS susceptible zone, probably modified the nucleus topology profile.⁹⁹ This phenomenon could be also connected with the uneven distribution of K+ and Na+ inside the cell, with Na+ being concentrated in the nucleus.¹⁰⁰ Thus, an increased nucleoplasm compaction, especially due to a high chromatin charge.^101–107 The modification of the nucleus chromaticity resulting from osmolyte’s concentration fast change^102–105 could have transiently displaced H1 histones^108–113 and brought other intervening nucleoproteins – such as with arginine-, lysine-, histidine-rich charges – and histones^{108,114–119} which made us here to recall our reader with the highly basophilia found in ODS12h oligodendrocytes with LM. Due to this kind of injurious ‘osmolyte’ challenge of hyponatremia fast restoration, would this phenomenon of tight repacking of chromatin and other nucleoplasm components associate with a self-protection mechanism for the genome out of an ‘epigenetic internal call’ that would salvage the genome of oligodendrocytes located around the epicenter of the myelinolysis zone with the possibility of a later reconstruction such as those hypothesized as in^{85,87,119–125}? In those important publications, only diagrammatic schemas have been evoked without imagery about reorganization of histones. This hyperchromatic morphology should correspond to a steep reduction or even stoppage of most nucleus transcription and translation activities, such as found in poisoning or cytotoxic influences or in natural preservation mechanism (hibernation) and corresponds to a low or even stoppage of some transcription and translation activities.^{15–18,101–105,117–119} Other techniques for molecular probe immunofluorescence should be welcome in the future along with electron microscopy to further explain such nucleus changes and cell compliance. Adaptive strategy to avoid cell death in murine ODS thalamus seemed to have been a success because, among the population of oligodendrocytes surveyed, one has encountered no more than one cell demise for more dozens of fields investigated, counting more than a total of 50 oligodendrocytes and, if any cell was undergoing injury, the necroptosis maker was only found, not that for apoptosis.^15,16 However, the hypothesis of epigenetic phenomenon cannot be dismissed until the gain of further data on these cells. The oligodendrocyte chromatin condensation as well as filled interchromatin zones with interchromatin granules grouping retrieved later all organelles and ribosomes found 36 hours later, when the oligodendrocytes appeared almost as the intact NN type. This signified some reestablishment of many cell’s functions (see here following paragraphs 4.c4 and 4.c5).

Figure 17. TEM of Interfibrillar oligodendrocytes of the ODS48h murine thalamus revealing an active, euchromatic aspect of organelles, including smooth and rough endoplasmic reticulum and large Golgi apparatus (G). In both A and B, the huge nucleus, adjacent to cross-, oblique and parasagittal axonal sections; astrocyte (As), and nerve cell body (N) with small damaged axon’s myelin (stars). In B: cell adjacent to a few remyelinated narrow axons (plain white arrows) with imperfections or other damages (stars); thickened contacts with astrocyte (As) (open white arrows), detected by cytoskeletal glial fibrillar proteins and glycogen storages (small white arrows). C: Enlarged part of an oligodendrocyte cytoplasm revealing abundant microtubules and cytoskeleton amongst polyribosomes, RER, and Golgi apparatus (G)

4.c2. The primary cilium

Discovered Zimmerman in epithelia and ducts of glands more than a century ago by,¹²⁷ this ‘rudimentary cilium’ or ‘solitary’ cilium was ultimately termed ‘primary cilium’ and its proteome has been deciphered.¹²⁸ It can be found in all epithelia and cultivated cell types of mammals, including those from mesenchyme-derived cells! This peculiar cell appendage has been the topic of innumerable studies and surveys in normal and pathology cells and tissues.^129–133 Adapted as a sensing device, contrarily to the typical cilium appendage, it lacks a central pair of microtubules and is non-motile. A superb 3-D reconstruction was reported by Sun and collaborators, using serial section electron microscope tomography.¹³⁴ Result of those studies, the detection in embryonal or developmental tissue cells¹³⁵ and, in some differentiated cells of lining apical surfaces was found to correspond to some mechanosensory (flow sensor), osmosensor wherever some fluid or flow control influence functions.^130,133 Thus, dysgenesis and mutations in the cilium-associated structure and membrane signaling proteins favor insensitivity and growth anomalies such as polycystic kidney disease (PKD), Bardet-Biedl syndrome (BBS) and/or other disorders as modulator function of tissue differentiation for the passageways where it is positioned.^{131–134,136} It is a vanilloid receptor-related osmotically activated channel.^136–138 As soon as normal differentiation happened, it usually disappeared, such as when interphase appeared with differentiation out of the cell cycle.^137,138 This trend toward cell cycle can reflect the arl13b marker found and the rare mitoses encountered after ODS as shown, with some potential for differentiation and reactivated myelination around the worse damaged ODS epicenter.¹⁷

Figure 18. Diagrammatic representation of an ODS oligodendrocyte primary cilium intracellular pocket formation above a centriole base where a fascicle axonemal microtubule is erected. The Golgi apparatus, adjacent to the nucleus (N) produces endocytic vesicles where an accretion process initiated around its shaft’s zone constructs a growing envelope and autophagosomes, with a contrasted content, as illustrated by micrographs in Figures 8A-13

4.c3. The oligodendrocyte primary cilium

When present in the mature CNS through fine structure of human neurosurgical biopsies and in experimental models, especially in vitro, this abnormal reappearance from development status and persistence can often be associated with a poor outcome as progression of a tumoral cases,^{126,133,135,138–140} such as glioblastomas,^140–145 astrocytomas,¹⁴⁶ medulloblastomas.^147–149 Some of these studies also showed that the primary cilium signaling in the pontine postnatal brain in excess would have suppressed macrophage and some astrocyte interactions with the oligodendrocyte remyelination, and thus, tumorigenesis [126, 131. 141, 143, 149]. In ODS syndrome, the sudden perturbations in the natremia may have caused other critical factors and/or osmolytes (amino-acids, myo-inositol, iron, etc.) transcytosis and effusion alterations through the BBB components and the intercellular CNS parenchyma or neuropil milieu^,15,16,19 that would modify some of the astrocyte’s functions linked with those of the joined oligodendrocytes.^15–17,19 One can hypothesize that the highly demanding metabolism of the oligodendrocytes^79–87 made them to undergo some self-protective genome mechanism, dodging death. It meant that, as viewed with fine structure, before 12 hours into the reestablishment of natriuremia, an ‘adaptative’ hyperchromaticity has been required not only to protect the genome but, with restoration of osmolarity, a series of signal pathways for ‘survival transduction’ have responded and, with those found in developmental autocrine (i.e. JAK/STAT along with interleukins factors)^150–153 interactively balanced others factors (Platelet-derived Growth Factor (PGDF), Brain-derived Neurotrophic Factor [BNDF] and/or Ciliary Neurotrophic factor [CNTF].^{64,67,153–159} It is remarkable that CNF and receptor conveyed cytoprotective effects after release from adult glial cells by some mechanism induced by injury¹⁵⁸ and could therefore heal these ‘injured’ oligodendrocytes to survive ODS or contribute to assist in their resilience without enduring cell death but a few, as noted before, especially in the myelinolysis EPM epicenter.^15,16

The detection of a primary cilium in mature murine oligodendrocytes located within and the edge of the demyelinated region of the brain thalamus after the 12^th hour after fast restoration of natriuremia is thus not fortuitous. For this cell appendage has not been found in vivo in mature CNS, only in vitro, where growth factor’s adjuvants have been used.⁵² Less than 12 hours post sodium osmolyte rebalanced, series of developmental genes installing a proteome capable of in vivo formation of a primary cilium were reawakened. This primary cilium occurred accompanied by Sonic hedgehog (Shh) and its expressed associated pathway.^160–175 These signals can favor a restoration of myelination functions by rejuvenation of the local, injured mature oligodendrocytes, including in human oligodendrocytes in vitro^52,168–170 because Hedgehog can relay Smoothened (Smo), another transmembrane receptor for embryonic development and adult tissue homeostasis. A balance between both signals, due of Patched1 (Ptc1) is a tumor suppressor that inactivates or modulates excessive signals between both Shh and Smo that localize on the primary cilium membrane.^161,162,164 Those transduction signals are currently being further studied throughout other laboratories and awaited more information. Incidentally, cholesterol building into the cilium lining shaft membrane is also regulated by Hh signaling.^{46–51,163,171–176} Among one of the ARF GTPases LM markers, the arl13b label revealed comforted this signaling process expressed as one of its characteristic proteins doting those microtubule-associated out of the centrioles,^45–53 and mutated in Joubert syndrome and other ciliopathies.^177,178 The immunolabel arl13b well characterized a primary cilium, transient to be along a cell cycle mitosis but resuming into oligodendrocyte extension elongation and remyelination^45–51 as illustrated in Figure 8A.

4.c4. Intracellular vesicles: resolution of transcriptional and translational activities

Hedgehog (Hh) signal transduction pathway in mice (as well as other mammals) is favorable to and necessary for the development and patterning the intracellular transport of vesicles sorted from the Golgi apparatus [as reported in the previous paragraph with arl13b protein revelation^{45–53,172–182} and effecting the homeostatic turnover of ‘used’ molecular entities with autophagocytosis, using the endosome as shown later, as enlarged (in ODS48h of Fig 13C), collecting as lipofuscin bodies. This cell’s vesicles were shown in numbers contained in these ODS12h oligodendrocytes along with the primary cilium have been called by a misnomer ‘intraflagellar’ transport (IFT)’ – even though there is no ‘flagellum’ built – during this intracellular built up! We use ‘IFT’ here only to acknowledge previous important studies of these Golgi secretory intracellular transports of peculiar phospholipids^172–190 with a terminology that should be amended as ‘cilum-associated vesicles’ (CAV) to encompass those that are being built and those that contained recycled components. These intracellular CAV vesicles cannot be dispatched to the plasmalemma surface because either the restrained space caused by crowding of the neuropil and neighboring macroglia and nerve cell bodies for a final outwardly exhibition or because the ‘program’ for building has been arrested due to reestablishment of differentiation, as seen at the 36- hour stage afterward. A peculiar intracellular furrow surrounding the tip of a centriole noted in,^190–197 is similar to typical cilium, with a microtubule alignment fascicle bearing these adjacent CAV aka ‘IFT’ vesicles distributed with small size and eventually lined to build the cilium shaft and envelopment.^180–186 The shaft and pocket membrane contained heterogeneous electron dense fuzzy membrane or cargo content, as the ciliary membrane either building or retrieving and recycling ‘old’ parts by autophagocytosis. It is because a distinct phospholipid composition has made them with heterogeneous contrast; the inositol phosphates, phosphatidyl-inositol 4-phosphate [PI (4)P] enriched the entire ciliary membrane and phosphatidyl-inositol 4–5 di-phosphate [PI (4,5) P2] augmented the base of the cilium.^182–187 All happened as in recall of a development-like state, when Hh signals have managed oligodendrocyte CNS placement and differentiation,^{61–65,85,170,171,176–196} having an intracellular cilium, as if aiming at a sort of extracellular sensing device that would but cannot protrude due to somewhat still-compacted parenchyma.^163–165 It can either be dismantled or alternatively, the centrosome forms a spindle in cell cycle and, thus, such structure appears as a sort of ‘rejuvenation’ signal left of the oligodendrocyte precursor-like cells found at ODS48h after rebalanced osmolarity. Like any construction, cells needed an energy demand that could be still supplied by diffusion and exchanged with connexins with the adjacent, less damaged or even intact astrocytes away from ODS damages, where the BBB had remained [15–17,19; 150–151, 170–171]. Intercellular junctions between astrocytes, oligodendrocytes, and neurons, as found in control, undamaged areas and would allow ions, signal molecules and small size metabolites to be maintained and provided.^{75–84,198,199}

4.c5. The oligodendrocyte primary cilium is labeled with arl13b antibody

Primary cilia are highly conserved multifunctional cell organelles that extend from the cell membrane, there, a wide range of genetic disorders, collectively termed ciliopathies, is attributed to primary cilia dysfunction, such as the Joubert syndrome.^175,178 Another archetypical ciliopathy is the Bardet–Biedl syndrome (BBS) where patients displayed all symptoms associated with dysfunctional cilia as in several other ciliopathies. One can see that the primary cilium acts as a sensory organelle transmitting intra‐ and extracellular signals as well as transfer phospholipids thereby transducing various signaling pathways facilitated by the BBS proteins.¹⁷⁸ Other growing evidences suggest that cilia proteins also have alternative functions in ciliary independent mechanisms, which might be contributing to disease etiology such as the family of ARF G proteins associated with the regulations of ATPases involved in the intracellular transport,^{42–50,171–173} including that of ciliogenesis (i.e., basal bodies and microtubule built up.^180–186 Thus, label for arl13b was strongly labeled at ODS12h specifically along with the detection of a polar particle-like that expanded with elongation, adjacent to the satellite oligodendrocytes by further encompassing the growth cone out of the cell bodies.^174,179,180 This finding would comfort other data where arl13b protein would associate with regrowth and strongly underlined the axon hillocks reestablishment of microtubule network.^{182,187–196} One can now imply and consider that a progressive process of post ODS has been initiated, healing the myelinolytic zone, as soon as the restoration of osmotic [Na+] was made. This healing process can also be supported by some replicating events that were revealed as cell cycle mitosis got detected for some oligodendrocytes and reentry into interphase with resulted ‘twin’ poorly differentiated cells.^200–205

4.d. ODS oligodendrocytes 48 h after rebalancing natriuremia

LM aspects of the p25alpha immunolabeling of the thalamus sections showed oligodendrocytes collected 36 hours after those of the previous group. They seemed to have not much changed in staining pattern. However, markers of the same oligodendrocytes still showed a loss in the same thalamus zones corroborated by a loss of APC immunoreactivity.^15,16 If the LM observation verified the worst myelin loss of marker at 48 hr post-correction with myelinolysis shown [(Fig 3) as in^15,16, there seemed to be a sort of damage gradient where some oligodendrocytes seemed to demonstrate strong p25alpha label and, as noted in ultrastructure, astrocytes joined these oligodendrocytes found in the typical panglial syncytium (Fig 5A-B) and,^58–62,198 as viewed in our Figures 13A-C -15A-B. These labeled cells at the edge of myelinolysis must have some tubulin organization^{86,87,199–202} as well as other small labeled cell or parts appeared between the large unstained nerve cell bodies. Due to the LM limited resolution it was thus essential to verify with ultrastructure the fine structure of this region. Meanwhile, finding replicating cells can be an outcome of restoring centriolar bodies and as rare scattered mitosis happened, their offspring can be ‘twin’ oligodendrocytes, even associated with astrocytes or neurons, initiating some remyelination.

With fine structure, none of the ODS48h oligodendrocytes revealed a primary cilium or its remnants and no autophagosomes were found in them. This finding and both labels with arl13b and p25alpha protein expressions indicated that microtubules are again organized out of the centrosome’s organelles.^{86,87,182,187,190,202–205} It also meant that a fast intracellular abrogation of this primary cilium structure that was viewed as an internal appendage in the mature CNS, with a transient existence. Would it be that it has been part of a mature status of ‘self-defence’ against the osmo-stress whose existence, short-lived, was related with a sensing of osmotic change and/or to undergo a sort of temporary ‘embryonal’ status^205–209 where myelin Hh-dependent signals became implemented.^138,169,170 This oligodendrocyte structure change could have occurred as a way to the cell directed toward differentiation similarly to those of interphase that revoked the tubulin huge expression organized for excess of mitosis of the cell cycle,^{144,166,170–175} circumventing immaturity^202–205 and its possible hedging toward pathologic anomalies evoked above.^{139–144,202} Rare mitosis found in this thalamus further verified that in the post-chronic hyponatremia stage healing oligodendrocytes toward remyelination prospect. Altogether, all these morphologic changes suggested some apparent myelin reconstruction even though, as noted above, some local, adjacent axonal defects were still detectable (swollen wrapping) with some small diameter axons that were completely surrounded by the oligodendrocyte perikaryon cytoplasm containing numerous microtubular profiles. Coincidentally, the adjacent neuropil revealed several evident axo-dendritic synapses.

Like in most maturing cells involved with early growth and differentiation, the finding of an euchromatic nucleus and active nucleoli in these oligodendrocytes with perikaryal display of organelles that included a Golgi apparatus, numerous sections of microtubules among many small cisterns of endoplasmic reticulum, whether smooth (SER) or rough (RER) were validating functional activity. In the same ODS48h oligodendrocytes, parts of RER encompassed long, straggling parts, spreading out the nuclear outer envelope and membrane extensions. Their structural and functional origin as outbursts from the perinuclear space is a reminder of their RER extension or origin.^206–209 Some of the mitochondria profiles of many of these cells remained with ‘scars’ earlier caused by osmolyte readjustment associated with ODS as shown in other cells of the macroglia. Furthermore, at this stage of ODS, astrocytes that contribute to the development of oligodendrocytes as well as myelination and a remyelination process have been noted to still maintain, not without intrinsic defects, intercellular connections with the oligodendrocytes.^15–17 and some myelinated axons – even though with some evident remaining damages – have again been associated with the apparently healed oligodendrocytes. We can also confirm that part of the unstained aspects in the myelinolysis zone were caused by intercellular spaces left from myelin debris as in a sort of ‘liquefaction’, described by LM histopathology, distancing neuropil components, and distal extensions of astrocytes, out of clasmatodendrosis^16,17,43 while neuron found in demyelinated or adjacent to them, with the oligodendrocytes described here, had undergone a Wallerian type of degeneration, preserving the nerve cell bodies¹⁸ and also retrieved transcription and translation activities. In view of the fine structures data surveyed in this report that included mainly the outskirts of the myelinolysis brain thalamus sections among a zone where demyelinated remnants caused small to large intercellular gaps, many oligodendrocytes, at the stages of ODS investigated, can demonstrate resilience from osmo-stress and reactivated transcriptional and translational activities.

Conclusions

Following ODS with demyelination of the murine ventral posterolateral (VPL) and ventral posteromedial (VPM) thalamic regions (the ventral posterior nucleus), a unique pattern of their oligodendrocyte morphologic changes have been illustrated, confirming some of the sequential alterations found with LM, that is, changes and loss of molecular markers for myelin occurring during and after 12 h and 48 h post rebalancing the natremia.^69,70 The search for CNS myelin repairs has been called previously.^210,212 Twelve hours after the fast restoration of the [Na+] the induction of developmental gene transcriptions resulted in a transient proteome associated with the phenotypic primary cilium (schematized in Figure 18). Found uniquely in development and, unregulated, in diseases out of the embryonal epithelium of many organs and it has been only found once in oligodendrocytes after cultivation.^52–54 In another word, this primary cilium is a sign of remyelination as the so-called ‘regeneration of the CNS’ as in the spinal cord.^212,213 Therefore, mature mouse CNS oligodendrocytes resilience would confirm that an epigenetic adaptation (not yet verified) has been aroused in order to eventually allow repair the extrapontine CNS injuries, instead of inviting for the immigration of oligodendrocyte precursor cells far out of the demyelination region.^210–216 Could this be as fast as in zebrafish, a model where the biological clock is evidently speedier than that of the mammal, influenced by aging?^217,218

Clinical considerations

This murine investigation model would suggest that a longer time lapse into ODS repairs could be investigated to understand the clinical resolution of such regional CNS neuropathology^1–18,219 because similar human ODS clinical cases remained with some concerns even if for most patients the prognosis is generally favorable.^7,220–222 However, a recent retrospective analysis indicated that, post hospitalization, 1 out of 6 ODS patients had died 6 months later, due to other underlying disease states.²²³ Accordingly, can astrocytes restore enough as parts of the blood-brain barrier components along with the repairing nerve axon’s extensions to return to standard functions?²²⁴ The astrocytes appeared the first to ‘alarm’ or keyed to trigger the oligodendrocyte changes with demyelination damages while the blood-brain barrier has been breached in ODS.¹⁷ Considering the latest information about demyelinating defects and some remyelinating potentials illustrated here one still remain to know whether with delayed time after ODS damages, a sort of modus vivendi between astrocytes, oligodendrocytes and neurons signals in making remyelination to return ODS thalamus or other CNS damaged regions completely able to retrieve standard structures and functions.

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Acknowledgments

This ultrastructural study was performed by JG, JB with CN and JB in the Electron Microscope facility of the” Plateforme Technologique Morphologie - Imagerie” of the Université de Namur with the technical assistance of Corry Charlier and Valérie Suain.

Additional information

Funding

This work was supported by grants T.0023.15 from the Belgium “Fonds de la Recherche Scientifique Médicale” and the Belgian Foundation “Recherche Alzheimer/Stichting Alheimer Onderzoek (14001; Fund Aline) to JPB. St George’s University School of Medicine, Newcastle upon Tyne, U.K. defrayed the Open Access page charge costs.