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Dromedary vimentin hypothalamo-neurohypophysal system expression may help adapt to severe arid environment.

Introduction

Intermediate filaments (IFs) participate in key mechanical functions and play a role in preventing excess large cell stretch. As such they are considered "safety belts of cells" because they make them tolerant to a variety of injuries such as mechanical stress, heat exposure, viruses, toxins, apoptosis inducing ligands [4,23]. Vimentin (57kDa, 7-11nm filaments), the most widely distributed type of IFs, is expressed in immature and dynamic cell conditions. For example, constitutive of mature neurons and glial cells, it is also associated with immature glia in the developing brain [18,2]. Actually, vimentin emerges as an organizer of a number of critical proteins involved in attachment, migration, and cell signaling because of its structure-property relationships. Thus, rather compliant at low strain, vimentin filament becomes much stiffer at high strain [11]. The hypothalamo-neurohypophysial system (HNS) is a dynamic neurosecretory system responsible for the production and release of vasopressin (VP) and oxytocin (OX) in response to water regulatory and cardiovascular challenges, and to the events of late pregnancy, parturition and lactation [21]. These two neuropeptides are synthesized in magnocellular neurons (MCNs) of supraoptic (SO) and paraventricular (PV) hypothalamic nuclei and stored and released on their terminals endings at the nervous lobe of the pituitary gland. VP and OX neurons often lie adjacent to one another, separated only by fine astrocytic processes. Neurosecretory terminals are engulfed by neural lobe astrocytes (pituicytes) positioned at the basal lamina that separates them from the fenestrated capillary [7]. With chronic physiological stimulations such as dehydration and lactation, the demand for these hormones is above basal level and a coordinated astrocytic withdrawal between the magnocellular somata, release of engulfed axons and synaptic reorganization takes place. This reduction in astrocyte surface density modifies glutamate removal and extracellular ionic homeostasis and results in a more efficient excitability of MCNs that enhances VP and OX release in the neural lobe capillary [7,9]. In fact, these neuronal-glial rearrangements represent the functional plasticity in which the increased cytoskeletal and adhesion molecules expression is engaged in the HNS responsiveness.

Research on dehydration HNS adaptation has been mostly developed in experimental rodent models receiving a stimulus, like a drinkable hypertonic NaCl solution, to mimic chronic severe water deprivation. In these conditions the structural HNS plasticity involves neuronal/glial cell interactions and correlates with the altered functional properties. However, some specific organization that efficiently responds to a natural chronic dehydration may not be present in these models. Because of that, the dromedary can be considered an animal of choice due to its unique cyclic adaptation to a strong arid biotope. In nature, this animal drinks every day in summer and 7 to 10 days in autumn and just every 4 to 6 weeks in winter [3]. Other authors reported that the period that the dromedary can support lack of water is between 15 to 20 days in summer and from 2 to 7 months in the other season of year [16]. In these conditions, this animal ensures a high degree of hydration through several mechanisms that compensate the reduced water availability and water loss. Thus, its daily diuresis, is reduced from 2.9-8.6 to 0.7-1.7 litre [1], a value lower than that observed in other desert mammals, This is primordially due to the renal action of vasopressin (VP) which develops 100 time faster than in bovine species [1]. To help understand this specific high adaptation to a prolonged dehydration, differences in its HNS organization can be present. Some authors have previously shown the presence of vimentin in the rodent HNS [2] linked to its capability to provide stability to the cells under adaptation. In the dromedary, differences in vimentin distribution and abundance may sustain the extremely large deformations required by the morphological neuronal and glial plasticity. To investigate this possibility, in this paper vimentin expression and distribution was characterized in the hypothalamus and hypophysis of adult male Camelus dromedarius as a factor involved in the cell remodeling.

Materiel and Methods

Brain Samples

Hypothalamus and hypophysis of 5 adult male one-humped camels (Camelus dromedarius) were collected after midnight from Golea slaughtherhouse (an Algerian saharien town 30.57[degrees]North longitude 20.87[degrees] East latitude) during the winter period.

After contention of the animals and their rapid decapitation, tissue preparation was adapted from human brain bank methods previously described by Mahy [12]. To this end, hypophysis and hypothalamus at the optic chiasm zone were quickly removed and cut into 1cm-thick coronal sections. Afterwards these sections were fixed in 10% neutral buffered formalin for 1 month. Interested parts of each brain were routinely dehydrated and embedded in paraffin using an automatic tissue processor. Serial sections (10 Lim) were mounted on gelatine-precoated slides.

Products

Products for immunostaining were obtained from DAKO, USA. The monoclonal antibody mouse anti-vimentin (Clone: VIM3B4, Isotype IgG1, kappa) was used at the 1/100 dilution.

Preparation of Tissues for Immunostaining

Deparaffinised sections of each hypothalamus and hypophysis were rehydrated and then three times rinsed in phosphate-buffered saline (PBS, pH 7.4). Endogenous peroxydase activity was blocked by preincubation of the tissue section in 0.3 hydrogen peroxide in methanol for 10 min at RT followed by three 10-minute washes in PBS. To permeabilize and block non-specific reactions; sections were placed in PBS with 3% normal goat serum and 0,5% Triton X100 for 1 hr at RT followed by three 10-minute washes in PBS.

Immunological Procedures

Pretreated sections were placed overnight at 4[degrees]C in an immunobuffer solution containing antibody against vimentin (in control sections the vimentin antibody was not included). The sections were washed and then immunostained with the DAKO LSAB2 biotinylated kit.

Sections were first incubated with biotinylated secondary antibody (DAKO ready to use LSAB2 Biotinylated Link, anti-mouse) for 2 hr at RT. There were then washed and reacted with a streptavidinperoxydase complex (Dako ready to use LSAB2 Streptavidin-HRP) for 2 hr at RT and the immunoreactivity was visualized using diaminobenzedin (DAKO LIQUID DAB) substrate. Sections were counterstained with haematoxylin, and then mounted with an aqueous mounting medium (DAKO Faramount Mounting Medium, Aqueous).

Observation and Photography of Sections

Sections were viewed with a light microscope for diagnostic use, LEICA DM LB2 and photographed by an adapted Canon power Shot S50 camera.

Results and Discussion

Vimentin Localisation in Hypothalamic Nuclei

Vimentin immunoreactivity (IR) was observed at the zone of the optic chiasm and the third ventricle of hypothalamus, in the supra optic nuclei (SO) and optic chiasm (OC) (Fig. 1A). At a magnified view, a strong reactivity is observed within the nucleus between magnocell neurons (MCN) (Fig. 1B,1C,1D). This vimentin-IR was localised in filamentous structures of astrocytes. These MCN were enriched with cytoplasm neurosecretory materiel surrounding cell nucleus and containing two to three nucleoli. Vimentin-IR astrocytes of the optic chiasm present a well-ramified filamentous shape resembling to a well-organised network (Fig. 1B). The glial components of the SO ventral zone that were vimentin-positive correspond to ventral glia limitans (Fig. 1E and 1F). This layer is mostly composed of astrocytes, and did not present extension of vimentin-positive glial processes through the nucleus.

The paraventricular nuclei (PV) is localized laterally to the third ventricle (3V) (Fig. 2A). Vimentin expression was observed along the 3V wall. At a higher magnification, the vimentin-IR was detected in glial cells, namely in ependymocytes that compose the wall of the 3V, and in astrocytes surrounding blood vessels (Fig. 2B). Some MCN from PV present vimentin-IR within their cytoplasm (Fig. 2C) associated with polarised cytoplasm granulations that may correspond to neurosecretory materiel (Fig. 2D) and (Fig. 2E). These MCN were associated with astrocytic processes and capillaries also positive for vimentin (Fig. 2E). Vimentinpositive astrocyte processes did not surround the MCN without granulation, and the adjacent capillaries were also poorly immunolabelled. In addition, the nuclei and nucleoli of both MCN presented in figures (2E) and (2F) showed different sizes.

Vimentin Localisation in Neurohypophysis

Compared to the intermediate lobe (IL), an intense vimentin-IR was observed in the hypophysis nervous lobe (NL) with the labelling of filamentous structures identified as pituicytes processes (Fig. 3A). The NL contained scattered islets composed by vascular systems through which liberation of neuropeptides is accomplished (Fig. 3B). Immunostain detected within these structures is weak comparing to the outside one , and the low signal was attributable to poor pituicyte processes abutting the capillaries of these islets (Fig. 3C).

Discussion

In our study, we have described for the first time the distribution of vimentin in the HNS of adult male dromedaries sacrified in winter season. In this area, several markers of activated MCN were observed, namely abundant cytosolic granulations and developed nucleolar materiel in the soma. Our major finding is the high vimentin-IR localized in neural, glial and vascular elements of the HNS. This wide expression and distribution of vimentin, an intermediate filament that can sustain extremely large cellular deformations, will provide stability to the HNS cells submitted to regular morphological adaptations caused by a seasonal severe arid biotope. The very high capacity of the HNS to properly process its dynamic state suggested by our results would explain, at least in part, the dromedary adaptation to summer and winter living saharian conditions.

In fact, structural plasticity has been already shown in MCN and glial cells of rat HNS by several authors, under chronic physiological stimulation such as lactation and dehydration [21]. This reorganisation is linked to glial cells retraction from their usual position between adjacent cell bodies of MCN [8].

In the NL, the structural plasticity is accompanied by a reorganisation of magnocellular terminals and adjacent pituicytes. These cells generally surround or enclose the terminals of MCN under basal conditions. In stimulated animals, the terminals become in direct contact with basal lamina, surrounding capillary vessels are enlarged and their number increased [24]. This enhancement of neurovascular contacts may be responsible for the facilitation of hormone release [8,22]. The proteins implicated in such plastic change, can be divided into two main groups, cytokeletal proteins and cell adhesion molecules [13].

We have observed in the dromedary SO, the presence of positive astrocytes between MCN. The VGL contains also a layer of vimentin-IR astrocytes in its coronal plane. In the rat, the VGL is composed of astrocyte cell bodies from which arises one long thick process that spans the SO, and several horizontally oriented processes that form a dense network, and a short process oriented towards the pia. These latter astrocytes are vimentin positive and suggest that the SO retains a certain degree of immaturity during adulthood, which may be linked to its well-known capacity to undergo neuronal-glial plasticity under physiological and experimental stimulation [2].

In the PV nuclei, vimentin, present in the MCN cytoplasm granulations, may be involved in the axonal transport, because vimentin filaments may be linked to microtubules and actin, and based on these networks, have also been associated with motor proteins [17]. Referred to the presence of cytosolic neurosecretory materiel observed in MCN of PV, some appeared clustered in one side of pericaryon, and others seemed to be moved to the distal part of the neurone (into NL). Only the vessels surrounding the first localisation were vimentin positive. New roles have been recently proposed for vimentin. Thus, through the regulation of integrin functions, vimentin would play key roles in adhesion [10] and would participate in the regulation of cell-cell contacts, especially in endothelial cells [6].

The functional significance of vimentin expression in ependymocytes of the 3V wall and adjacent astrocytes of PV may be to increase the cell flexibility in ionic (K+) clearance during the repeated electrical activation of MCN. In the neurohypophysis this protein is concentrated outside the vascular system seemingly dilated and enclosing several vessels. Its localisation is in pituicytes processes and may be in endothelial cells, might enhance the access of neuropeptides to blood stream by improving cell shape, as shown for vimentin in immature glia during development [15]. In fact, pituicytes make an active contribution to structural plasticity by either withdrawing or extending their cellular processes over the magnocellular terminals [24].

The physiological strategies that enable the dromedary to survive long periods with no access to water reflect a continuous stimulation of the HNS that secretes hormones implicated in water balance. Our results bring information on several aspects of neuronal activation.

Conclusion

The expression of vimentin in most glial elements of the HNS can be viewed as the "safety belt" that play a key role in preventing exceeding large cellular stress. Taking together, our data suggests a high capacity of the dromedary HNS to undergo neuro-anatomical changes through adult life derived at least in part from elevated vimentin expression in most cell types and the maintenance of a certain degree of immaturity. These aspects should help explain its adaptation to a severe arid environment.

Acknowledgements

The authors would like to thank Pr. Z.C. Amir (Anatomo-pathology service Mustapha Bacha hospital; Algiers) for her generous gift of vimentin antibody. Elaichi F and Behri K. for their help.

Fig 1: Vimentin localization in the supraoptic nuclei. Rostraly, the SO nucleus is localized on the dorsal and lateral zone of the OC. Vimentin immunodetection was observed in the SO and in OC (A,B) and between MCN (C) and (D). The cytoplasm of magnocells was fully occupied by neurosecretory material(C); some presented one to two nucleoli and others three, reflecting a high secretory activity (arrows) (D). Between neurons, filamentous structures positive for vimentin; may correspond to astrocytic processes (arrowheads) (D).The VGL composed of astrocyts appeared positively marked in the close proximity of the ventro-rostral part of SO nucleus (E,F) . Ast=astrocyte, MCN= magnocell neuron, OC= optic chiasm, SO= supraoptic nucleus, VGL= ventral glial limitans. Scale bars= 200Lim in (A); 100 Lim in (B), 10[micro]m in (C) and in (D); 20 [micro]m in (E); 50 [micro]m in (F).

Fig. 2. Vimentin localization in the paraventricular nuclei. The PV nucleus (arrow) (A) is localized laterally to the wall of 3 V. Vimentin immunodetection in this zone; was very intense along the 3V wall (arrowheads) (A). Positive-IR is observed in ependymocytes forming the wall of 3V, astrocytes surrounding blood vessels (arrow) and scattered ones (arrowheads) (B). Most of MCN of the PV presented a positive cytoplasm reaction (arrows) (C), but some MCN did not contain a positive-IR (arrowheads) (C). The immunolabel seemed to be associated with a granular neurosecretory materiel (star) concentrated in one side of pericaryon that could be the initial part of axon (D). Vimentin was also present in astrocyte processes and/or capillary wall (arrowheads) (D). MNC deprived of granulations in their cytoplasm did not express vimentin and were surrounded by very few vimentin-positve structures like capillaries (arrowheads) (E). PV= paraventricular nuclei, 3V= third ventricle. Scale bars= 50Lm in (A); 5 [micro]m in (B); 10 Lim in (C); 100 Lim in (D) and in (E).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Fig 3: Vimentin localization in neurohypophysis. Compared to the (IL), an intense vimentin immunoreactivity was observed in the NL (A). In (A) arrowheads indicate a complex vascular system (scattered in NL as islets) through which liberation of neuropeptides is enhanced. Vimentin immunoreactivity was essentially concentrated outside these vascular system islets; with abundant processes abutting these lasts (B). (C) Detailed observation of an islet, with low inside vimentin labeling (arrowheads). Arrow shows a capillary containing red blood cells. IL= intermediate lobe of hypophysis, NL= nervous lobe of hypophysis. Scale bars= 5[micro]m in (A); 10 [micro]m in (B); 100 [micro]m in (c).

[FIGURE 3 OMITTED]

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(1) Djazouli Alim Fatma Zohra, (2) Lebaili Nemcha, (3) Mahy Nicole

(1.) University SAAD Dahleb; Fac. Agro-Veterinary Science; B.P. 270 route de Soumaa Blida; Algeria.

(2.) Ecole Normale Superieure, Laboratoire de Physiologie Animale; E.N.S de Kouba Bachir El Ibrahimi, B.P 92, 16050 Algeria.

(3.) University Barcelona; Unit of Biochemistry, Sch. Medicine-IDIBAPS, C. Casanova, 143 Barcelona, 08036, Spain.

Djazouli Alim Fatma Zohra, Lebaili Nemcha, Mahy Nicole,Dromedary vimentin hypothalamoneurohypophysal system expression may help adapt to severe arid environment Adv. in Nat. Appl. Sci., C(C): CC-CC, 2010
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Title Annotation:Original Article
Author:Zohra, Djazouli Alim Fatma; Nemcha, Lebaili; Nicole, Mahy
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:6ALGE
Date:May 1, 2010
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