Pericytes of the Brain in Demyelinating Conditions

Abstract

The pericytes play a very important role in the central nervous system (CNS), concerning the formation of the functional neurovascular unit, serving as a substantial component in the development and maintenance of the stability of the blood-brain barrier (BBB). Besides, as pluripotent cells of neuroectodermal origin, the pericytes participate in autoimmune reactions and modulations, controlling the penetration of immune cells via BBB and playing an active role in lymphocytic trafficking and functional regulation, via cytokine secretion and activation. In demyelinating conditions, they participate in the restoration of the myelin sheath by modulating oligodendrocytes and stimulating the differentiation of oligodendrocyte progenitors. In the experimental model of allergic encephalomyelitis (EAE), electron microscopy reveals the proliferation and the morphological alterations of the pericytes as well as their interactions with endothelial cells and astrocytes, thus underlining the crucial role that pericytes play in the integrity of the BBB and the immune reactions of the CNS.

1. Introduction

Multiple sclerosis (MS) is among the most enigmatic disorders of the central nervous system, affecting a substantial number of patients, at any age from childhood to senility, inducing a large spectrum of physical and mental disability in a considerable number of them, with a high prevalence in Europe and North America [1].

The clinical diagnosis of multiple sclerosis is not always an easy task, due to the polymorphic and multidimensional pattern of the clinical manifestations of the disease, which might be associated with other disorders.

The phenomena and the severity of the disease would be evaluated based on the criteria of the expanded disability status scale (EDSS) [2].

The pathogenesis of MS, which has been considered as a chronic immune-mediated disorder of the central nervous system (CNS) [3], has to be further clarified, although some risk factors such as genetic predisposition, viral, bacterial, or parasitic infections [4], climatic, environmental, and dietary factors [5], head trauma, and physical or psychological distress may play a substantial role in the puzzling etiological background of the disease.

Besides, the multidimensional underlying pathological mechanisms of multiple sclerosis, involving numerous cell interactions, molecular reactions, and activation of autoimmune responses via a multitude of signaling factors, result in inflammatory infiltration, demyelination, gliosis, and axonal damage, which compose a very complicated labyrinthine pattern, causing a reasonable difficulty in the effectivity of any targeted therapeutical approach of the disease [6].

Among the many cellular components, which participate actively in the process of demyelination and remyelination, during the continuous neuropathological alterations and interactions in the brain and the spinal cord, during the long course of multiple sclerosis, the pericytes being heterogeneous cells [7] described by Rouget, originally called Rouget cells [8] and named pericytes by Zimmermann [9], seem to play a substantial role at any stage of the disease.

It is well known that brain pericytes are pluripotent progenitor cells of neuroectodermal origin [10, 11], which are located mostly around the blood vessels (peri, περι = around) and serve as substantial components of the blood-brain barrier (BBB), being in direct contact with the endothelial cells, sharing a common basement membrane with them, and developing many functional interactions with endothelial cells, astrocytes, perivascular microglia, and macrophages [12].

It is important that the pericytes contribute to the formation of the functional neurovascular unit (NVU), which is composed of endothelial cells, pericytes, astrocytes, and neurons, and serve as a crucial structure for the integrity and functional stability of the central nervous system [13, 14]. The pericytes participate also in the development of the wall of small vessels, such as pre-capillary arterioles, capillaries, and post-capillary venules, enveloping the endothelium and being separated from them by a basement membrane (BM). Over most, the fact that the pericytes play a crucial role in the function of the blood-brain barrier [15] is of particular importance, particularly in the development of the tight junctions and in the vesicle trafficking in the endothelial cells, controlling the permeability of the BBB and participating effectively in its reconstruction and remodeling, in cases of anatomical disruption or functional decline, contributing therefore essentially in the stability of brain homeostasis [16].

A substantial body of evidence revealed that inducible pericyte knockdown in experimental animals resulted in disruption of the blood-brain barrier and rapid loss of neurons [17, 18].

It is important that the pericytes do frequently migrate in the neuropile space and even proliferate into different cellular types participating in a multitude of cell interactions [19].

Besides, pericytes participate in autoimmune reactions and modulations, mediating the neuroinflammation [19], and controlling the penetration of immune cells via BBB, playing an active role in lymphocytic trafficking and functional regulation via cytokine secretion and activation [20, 21, 22], and eventually contributing in glial scar formation [23].

The fact that pericytes are involved in the remyelination of the CNS, by modulating oligodendrocytes and stimulating the differentiation of oligodendrocyte progenitors [24], is of substantial validity.

The density of the pericytes varies from tissue to tissue, being the highest in the CNS, apart from the retina [25]. However, their number is not definitely stabilized, given that they could differentiate into other cell types, including glial cells and neurons under various conditions, in reaction to tissue injury [26].

For a further observation and detailed analysis of the gradual neuropathological phenomena and the cellular interactions, which occur in multiple sclerosis, animal models have been created by active immunization of susceptible recipients [27]. Among them, the experimental allergic encephalomyelitis (EAE) is the most frequently used animal model [27], which has been induced in genetically susceptible animals such as rats, mice, guinea pigs, rabbits, and monkeys by injecting compounds that would stimulate the immune system, resulting in developing inflammatory perivascular infiltrates in the CNS [28].

In the majority of the experimental models, the injected immunogenic factor is derived from CNS proteins such as myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG). The injected animals may develop neurological manifestations due to creation of inflammatory foci and demyelination in random areas of the CNS, in analogy to MS [29, 30].

Among the cellular components, the pericytes [31], the microglial cells, and the perivascular macrophages (PVM) [32] may play a substantial role in mediating neuroinflammation in the CNS in the EAE, as well as in MS and other autoimmune neurological conditions [32, 33, 34].

In the present study, we attempted to study the ultrastructural alterations of the pericytes around the capillaries and the venules of the brain in animals, which developed experimental allergic encephalomyelitis, knowing that there are some substantial limitations, due to the different pathogenetic mechanisms of the EAE, in correlation with MS [35].

2. Material and methods

2.1 Material

Sixty adult Lewis rats (AgB1) of both sexes (30 male and 30 female animals), of 150–200 mean body weight, were immunized by slow injection in their hind footpads of 50 μm of guinea pig myelin basic protein, emulsified in Freund’s complete adjuvant.

All the rats were clinically examined and scored daily following the immunization. The first clinical findings appeared between the 10th and 12th day following the injection. On the 18th day after the immunization, all the clinical manifestations of the animals were quantified and scored based on a 0–5 disease severity scale, where 0 means no clinical findings, 1 means loss of the tone of the nail, 2 means weakness of the hind limb, 3 means paralysis of the hind limb, 4 means paralysis of the hind limb and severe weakness or paralysis of the forelimb, and 5 means expiring condition or death [36, 37].

From the 60 immunized animals according to the final clinical evaluation, 2 of them were scored 0, 12 were scored 1, 10 were scored 2, 22 were scored 3, and 14 were scored 4. No one died.

Then, under ether anesthesia, all the rats were sacrificed, by perfusion with 200 ml buffer solution (buffered physiologic saline) followed by 250 ml οf Sotelo fixing solution [38] containing 2.5% glutaraldehyde, 1% paraformaldehyde in 0.2 cacodylate buffer, adjusted at pΗ 7.35. For the perfusion, a Holter pump (flow 25 ΗΡΜ) was used.

After the fixation, the skull of each animal was opened as well as the spinal canal, and the brain and the spinal cord were quickly removed and immersed in newly prepared Sotelo solution at 4°C.

2.2 Method

Coronal sections of the brain hemispheres were performed. The brain stem, the cerebellum, and the spinal cord were cut into sections of 2 mm thickness. Samples were taken under a dissecting microscope and immediately processed for electron microscopy.

All the specimens were immersed in newly prepared Sotelo fixing solution, for 3 h, then they were postfixed in 1% of osmium oxide for 30 min at room temperature and dehydrated in graded alcohol solutions and propylene oxide. After the dehydration, the specimens were embedded in Araldite mixture.

Semi-thin sections were performed on a Porter-Blum microtome and stained with 1% toluidine blue. Thin sections of silver-gray inference color were cut in a Reichert ultratome, mounted on bare 400 mesh grids, contrasted, with uranyl acetate and lead citrate, and studied in an electron microscope Zeiss 9As.

Besides, multiple sections of the brain hemispheres, the brain stem, the cerebellum, and the spinal cord, were prepared for histological examination. Thus paraffin-embedded sections were stained with hematoxylin and eosin and were serially studied in the light microscope at a magnification of 25× and 100×.

The histological diagnosis of the experimental allergic encephalomyelitis was based on the identification and quantitation of the perivascular infiltrates of mononuclear cells and lymphocytes in the brain, the cerebellum, and the spinal cord.

3. Results

3.1 In light microscopy

The histological examination of the H and E stained sections revealed a substantial number of perivenular and pericapillary infiltrations in the brain hemispheres, the brain stem, and the cerebellum of the animals, with the greatest amount of infiltrations seen in animals, which were scored 4 and 5. The spinal cord was seriously involved showing the highest number of perivascular infiltrates in all animals. No infiltrations were observed in animals scored 0.

The semi-thin sections of Araldite embedded tissue, which were studied in light microscopy revealed, besides the perivascular infiltrates, alterations of the myelin sheath of the myelinated axons in the brain hemispheres, the cerebellum, and extensively in the spinal cord in animals scored 4 and 5.

3.2 In electron microscopy

By electron microscopy, the pericytes were seen in the wall of the brain capillaries, around the endothelial cells (Figure 1). They are characterized by their large round or ovoid nucleus, with rough distribution of the chromatin, the plenty of mitochondria and ribosomes perikaryon, and the basal lamina, which surrounds the cell body. They interacted with the endothelial cells, which create gap junction, surrounding them.

Figure 1.

Pericyte (P) in the wall of a brain capillary near the endothelial cell (E). Electron micrograph (mag. 35,000×).

A substantial proliferation of pericytes was noticed in the spinal cord and the cerebellum around the capillaries and the venules, escaping the basal lamina (Figure 2) particularly in animals scored 4 or 5. All of them extend long processes, on the one hand surrounding the wall of the blood capillaries and on the other approaching the astrocytes in the perivascular space.

Figure 2.

Pericyte escaping the basal membrane (bm) around a brain capillary. The mitochondrial alterations are obvious. Electron micrograph (mag.128,000×).

Morphologically, the majority of the pericytes and the endothelial cells demonstrated aggregations of many small mitochondria around the nucleus, dilatation of the cisternae of Golgi apparatus, and large lysosomes (Figures 2 and 3). The nucleus of the activated perivascular pericytes was mostly round or ovoid, distinguished clearly from the very elongated nuclei of the endothelial cells (Figure 4). The nucleus of the perivascular pericytes demonstrated, as a rule, a rough distribution of heterochromatin in the periphery. The perikaryon included large number of small round mitochondria, with fragmentation of the cristae in the majority of them. A substantial number of endothelial cells demonstrated dilatation or disruption of the tight junctions (Figure 5).

Figure 3.

Alterations of the mitochondria and dilatation of the smooth endoplasmic reticulum (ser) in an endothelial cell of the wall of a brain capillary. Electron micrograph (mag. 128,000×).

Figure 4.

An endothelial cell of a brain capillary, showing abundant peripheral accumulation of heterochromatin and disruption of the tight junction (Tj). Electron micrograph (mag. 35,000×).

Figure 5.

Disruption of the tight junctions (Tj) of a brain capillary. Electron micrograph (mag. 128,000×).

Many capillaries showed marked perivascular edema and accumulation of lymphocytes and monocytes. It was noticed that pericytes in the neuropile space were intermixed with astrocytic processes (Figure 6).

Figure 6.

Pericytes (P) in the neuropile space around the endothelial cell (E) of a dilated brain capillary. There is a marked perivascular edema. Electron micrograph (mag. 35,000×).

A large number of pericytes demonstrated an increased number of pinocytotic vesicles, large lipid granules, and mitochondrial alterations, and marked dilatation of the cisternae of the smooth endoplasmic reticulum (Figures 2 and 3).

4. Discussion

Pericytes are polymorphic perivascular cells, which collaborate with the endothelial cells for the regulation of the blood–brain barrier’s permeability [39]. A substantial body of evidence, derived from morphometric observations in light and electron microscope, revealed that the ratio of pericytes to endothelial cells in the majority of the structures of the central nervous system is approximately 1:1 [39]. However, not all of the perivascular cells are pericytes, given that some of them are macrophages or adventitia cells [40], presumably derived from the pericytes, which as pluripotent cells can generate other cell types, to maintain the brain homeostatic equilibrium [22].

In MS, the proliferation or the degeneration of the pericytes associated with dysfunction or disruption of the blood-brain barrier is one of the initial neuropathological phenomena [41], triggering a cascade of inflammatory reactions and cellular interactions.

In the model of the experimental allergic encephalomyelitis, alterations of the blood-brain barrier have been described in electron microscopy by many authors [42, 43, 44]. The role of the pericytes in inducing those alterations may be crucial, given that perivascular pericytes regulate endothelial transcytosis, which would increase the permeability of the blood-brain barrier [45].

In the model of pericyte-deficient mice, an increased expression of leukocyte adhesion molecules has been described in association with polarization defect of astrocyte end feet in the vessels of the brain, underlining the importance of the pericytes for the integrity of the blood-brain barrier [46].

On the contrary, the proliferation of the pericytes suggests that they participate in the immune reactions of the brain, a fact that is noticed and described also in multiple sclerosis [21]. It was noticed that the pathological alterations in experimental allergic encephalomyelitis mimic to some degree, in many aspects, the morphological alterations, which occur in multiple sclerosis [47, 48].

Many histological observations revealed that the morphology of the pericytes varies considerably in the various structures of the brain in normal and pathological conditions. Among other conditions, proliferation of pericytes was described in early cases of Alzheimer’s disease, associated with disruption of the BBB [49] as well as in traumatic brain injuries [50].

Although many markers have been used for the identification of pericytes in various conditions, none is unanimously accepted as the precise and definite one, given that pericytes retain the multipotential properties of stem cells [51] or express a macrophage-like function [52].

The proliferation of the pericytes around the capillaries and the venules in the central nervous system has been observed mostly at the initial stages of the inflammatory conditions, autoimmune reactions, and degeneration of the brain, given that as the process advances, the pericytes further migrate into the neuropile space, and the ratio between them and the endothelial cells declines consequently [53].

In many pathological conditions, the pericytes contribute to the restoration of the BBB substantially, either by their contact with the endothelial cells or through proper signaling [54, 55], a fact that is beneficial for the establishment of the brain homeostasis.

A reasonable therapeutic approach to multiple sclerosis may be attempted by enforcing the interactions between pericytes, endothelial cells, and astrocytes, which may result in the restoration of the blood-brain barrier [56, 57].

It would also be emphasized that the observation that activated pericytes may contribute substantially to the differentiation of the oligodendrocyte progenitors, enabling consequently the restoration of the myelin sheath, and the protection of the axons is of substantial importance for finding an escape from the labyrinth of the disease [58]. This novel role of pericytes may open new therapeutic horizons in the field of demyelinating conditions [59, 60], as a catharsis from the drama of multiple sclerosis.

5. Conclusions

1. Pericytes play a very important role in the formation and mentainance of the blood-brain barrier (BBB), as a substantial component of the neurovascular unit.

2. Pericytes participate actively in the autoimmune reactions of the central nervous system (CNS), having the capacity to interact with oligodendrocytes and astrocytes and even to generate other cell lines.

3. In the experimental model of multiple sclerosis (MS), the experimental allergic encephalomyelitis (EAE), the electron microscopy shows clearly the proliferation of the perivascular pericytes, their migration into neuropile space, their morphological alterations, and even their collaboration with endothelial cell, for the restoration of the disrupted BBB.

4. Activated pericytes may contribute to the differentiation of the oligodendrocyte progenitors, a fact that may enable the restoration of the myelin sheath and increase the axonal protection.

5. Therapeutic regimes protecting the pericytes in the early stages of demyelinating conditions may open new promising horizons in the treatment of multiple sclerosis.

Conflict of interest

The author declares no conflict of interest.