INTRODUCTION

Fibroblast-like cells play a central role in various diseases of different organs, including the synovial membranes of diseased joints (^{Kontoyiannis & Kollias, 2000}; ^{Mor et al., 2005}), the myocardium (^{Takemura et al., 1998}; ^{Hayakawa et al., 2003}; ^{Yano et al., 2005}), and the liver (^{Jodon de Villeroché & Brouty-Boyé, 2008}). These cells area mostly differentiated into different types, such as neurons, corneal cells, osteoblasts, chondrocytes, adipocytes, cardiomyocytes, hepatocytes, and pancreatic islet cells. Despite originating from a non-embryonic source, they express stem cell markers that are important for maintaining an undifferentiated state. This multipotent property makes these cells a promising therapeutic target (^{Dravida et al., 2005}; ^{Giovannini et al., 2008}). In rheumatoid arthritis, fibroblasts-like cells, called fibroblast-like synovial cells (FLS) stimulate the inflammatory process and directly attack the cartilage and bones. Moreover, FLS modulate the autoimmune response, and are crucial in the pathogenesis of rheumatoid arthritis (Kontoyiannis & Kollias; Mor et al.). Fibroblast reorganisation is associated with neoplastic progression, and is associated with the inflammatory process, and macrophage recruitment is associated with neoplasia (^{Coussens & Werb, 2002}; ^{Allinen et al., 2004}; ^{Amatangelo et al., 2005}).

Fibroblasts control local and distant angiogenesis mediated by bone marrow stem cells; however, they are transformed into invasive types and myofibroblasts (^{De Wever & Mareel 2003}; ^{Orimo et al., 2005}) within the stroma of invasive cancers of the breast, colon, ovaries, pancreas and prostate (^{Kalluri & Zeisberg, 2006}). After wound injury, fibroblasts are activated and help recruit mesenchymal stem cells that play a role in wound healing (^{McGee et al., 2013}). Cardiac fibroblasts are transformed into myo-fibroblasts, which are important in tissue contraction and repair after myocardial infarction (Takemura et al.; Yano et al.). Moreover, they can be converted into a myogenic phenotype, which may be helpful for the treatment or prevention of myocardial infarction (^{Salvatori et al., 1995}; ^{Wise et al., 1996}; ^{Etzion et al., 2002}; ^{Goldsmith et al., 2004}; ^{van Tuyn et al., 2007}). The biliary tract and hepatic epithelial cells arise from the portal tracts surrounding ducts, where fibroblasts are abundant (^{Crosby et al., 2001}; ^{Libbrecht et al., 2002}; Li et al., 2013; ^{Lemoinne et al., 2013}). In vitro studies using fibroblasts from different tissues showed that fibroblasts display distinct behaviour according to the anatomic site and the disease status (^{Chang et al., 2002}; ^{Parsonage et al., 2003}).

Endoneurial oedema is a prominent feature in most types of neuropathies (^{McManis et al., 1986}; ^{Olsson, 1989}; ^{Pedowitz et al., 1991}; ^{Gabreëls-Festen et al., 1993}; ^{Eaton et al., 1996}; ^{Maxfield et al., 1997}). It has a major role in reducing the endoneurial blood flow and subsequent degeneration of the nerve fibres (Maxfield et al.). During nerve regeneration, the endoneurial oedema subsides, leading to decreased endoneurial pressure and increased blood flow (^{Low et al., 1985}; Gonda et al., 2010; Kobashigawa et al., 2011). In diabetic neuropathy, the progressive nerve fibre loss may be attributed to the persistent nature of the endoneurial oedema (Low et al.). Endoneurial oedema accumulates primarily in the subperineurial and perivascular spaces (Pedowitz et al.; Gabreëls-Festen et al.). The reason for this localisation and fate of the accumulated endoneurial oedema in different types of neuropathies remains unclear. The question remains, do fibroblast-like cells have a role in nerve regeneration and the localisation or resolution of endoneurial oedema following nerve injury Therefore, the present study assessed endoneurial fibroblast-like cells and their possible role in the localisation and resolution of endoneurial oedema.

MATERIAL AND METHOD

Forty-two albino adult male Sprague- Dawley rats were used in this study, weighing 200 to 250 g and aged 3040 days. A total of 36 rats were initially anaesthetised by ether inhalation, followed by an intraperitoneal injection of penta-barbitone sodium (Sagatal; 60 mg/ml) at a dose of 30 mg/kg body weight. Anaesthesia was maintained during surgery by repeated intraperitoneal injection of pentabarbitone sodium. The left sural nerves were exposed and subjected to crush injury just distal to its origin from the tibial nerve using non-toothed forceps for 10 seconds. The right and left sural nerves of the remaining 6 rats were used as a control group. At set intervals ranging from 1 to 3 weeks after the nerve crush, 12 animals were re-anaesthetised initially by ether inhalation, and then weighed and injected with penta-barbitone sodium according to their weight. The crushed nerves were re-exposed and collected. A 2.5 mm segment at the site of the crush was discarded. The remaining nerve was segmented into 2.5 mm segments, fixed in 2.5 % glutaraldehyde then post-fixed in 2 % osmium tetroxide. The nerve segments were dehydrated in graded concentrations of ethanol and embedded in Araldite resin. Semithin sections of 1 µm were obtained and stained with toluidine blue in borax for light microscopy. Ultrathin sections of 0.07 µm were stained with uranyl acetate and lead citrate for electron microscopy. Sural nerve segments were also collected from the normal nerves and processed similarly.

Light Microscopic Study

Light Microscopic Study of Normal Sural Nerves. The endoneurium contained myelinated nerve fibres with different diameters and thicknesses of their myelin sheaths. There were few scattered endoneurial fibroblasts with short cytoplasmic processes throughout the endoneurium without special arrangement. The subperineurial space contained myelinated nerve fibres (Fig. 1A).

Light Microscopic Study of Crushed Nerves. One week after the crush injury, the endoneurium showed a significant increase of the number of endoneurial fibroblasts and fibroblasts-like cells with long cytoplasmic processes. Some were scattered in the endoneurium, whereas others surrounded the regenerating nerve fibres. Fibroblast-like cells surrounded the regenerating nerve fibres. There was an increase in degenerated myelin and endoneurial macrophages (Fig. 1B).

Two weeks after the crush injury, the flattened fibroblast-like cells were arranged in multiple cellular layers in the subperineurial space. The flattened cytoplasmic processes of the fibroblast-like cells were attached in the majority of layers, forming subperineurial barrier-like cellular sheets. Fibroblast-like cells also surrounded the regenerated nerve fibres, isolating them in special compartments. The spaces between the fibroblast-like cellular sheets and perineurium contained accumulated endoneurial oedema, in addition to some of the endoneurial macrophages carrying degenerated myelin (Fig. 1C).

Three weeks after the crush injury, the fibroblast-like cells were arranged in a nearly continuous cellular sheet in the subperineurial space, forming a cellular barrier-like structure. Other cells surrounded the regenerating groups of nerve fibres. The subperineurial space contained endoneurial oedema and endoneurial macrophages (Fig. 2).

Fig. 1A A normal sural nerve showing the perineurium (PER), endoneurium containing myelinated nerve fibres (long arrows), few scattered fibroblasts (arrow heads) and the endoneurial blood vessels (BV). B. One week after the crush, there are increased degenerated myelin (long arrows), increased number of endoneurial fibroblasts (short arrows). C. After two weeks of the crush injury, the endoneurial fibroblast-like cells form several cellular sheets in the subperineurial space (arrows) forming barrier-like sheets. O, subperineurial oedema, MG; macrophages, PER; perineurium, BV; blood vessels. X 400. 

Fig. 2 After three weeks after the crush injury, the endoneurial fibroblast-like cells form a nearly continuous cellular sheet in the subperineurial space (arrows) localizing the subperineurial oedema. PER; perineurium. X 400. 

Electron Microscopic Study

Normal Sural Nerves. The endoneurium showed myelinated and non-myelinated nerve fibres along with collagen bundles together with the endoneurial blood vessels. Endoneurial fibroblasts were few and scattered in the endoneurium, and a few had longer cytoplasmic processes. The subperineurial spaces were filled with collagen bundles and nerve fibres (Fig.3A).

Crushed Sural Nerve. One week after the crush injury, the endoneurium showed the accumulation of endoneurial oedema indicated by the wide separation of collagen fibrils. It showed an increased number of flattened endoneurial fibroblast-like cells, with long cytoplasmic processes that wrapped the regenerating unmyelinated or thinly myelinated nerve fibres, in most of the specimens. There were areas of contact between these cytoplasmic processes and the nearby endoneurial macrophages. Some of the endoneurial fibroblasts were flattened and fibroblast-like, whereas others appeared fusiform (Figs. 3B, C).

After two weeks after the crush, the endoneurial fibroblast-like cells were arranged in cellular layers in the subperineurial and around the regenerating nerve fibres, forming barrier-like cellular sheets (Figs. 4). Channel-like spaces appeared in the endoneurium. These spaces are lined by fibroblast-like cells and collagen bundles and contained degenerated myelin debris and accumulated endoneurial oedema. The channel-like spaces were continuous with the subperineurial space. The endoneurial macrophages had areas of contact with the endoneurial fibroblast-like cells.

The subperineurial barrier-like sheets were interrupted where the channel-like spaces communicated the subperineurial space and also where the endoneurial macrophages were located (Fig. 4).The endoneurium was divided into areas of regenerated nerve fibres surrounded by the cytoplasmic processes of the fibroblast-like cells (Fig. 4).

During the third week after the crush injury, the endoneurial fibroblast-like cells were apparently decreased in number, with the disappearance of most of the endoneurial channel-like spaces. The fibroblast-like cells wrapped the regenerating nerve fibres in most of the specimens and formed single subperineurial cellular sheet (Fig. 5).

Fig.3. A Normal sural nerve with myelinated (m), unmyelinated (u) nerve fibres and collagen bundles (C) in the subperineurial (SUB) space. PER, perineurium. Scalebar 1 µm. B. One week after crush injury, the endoneurial fibroblast (F) and the endoneurial fibroblastlike cells were increased in the subperineurial space among the regenerating nerve fibres, there is accumulation of endoneurial oedema (O). Scale bar, 5 µm. C. Endoneurial fibroblasts with long cytoplasmic processes (pr), with cytoplasmic rough endoplasmic reticulum (rER) and mitochondria (M) that partially surrounded the regenerating nerve fibres (R) and have areas of contacts with the endoneurial macrophage (MG), one week after the crush. Scale bar, 2 µm. 

Fig. 4 The sural nerve after 2 weeks of the crush injury shows the endoneurial channel-like spaces (CH) lined by fibroblast-like cells (arrows) and collagen bundles (C). They contain degenerated myelin and endoneurial macrophages (MG), and some are continuous with the subperineurial (Figs. 5A-5D). Fig. 5D shows subperineurial cellular barrier-like sheets of fibroblast-like cells (arrow heads). PER, perineurium. Scale bars 10 µm, 2 µm, 10 µm and 10 µm, respectively. 

Fig. 5 The sural nerve during the third week after crush injury showing the endoneurial fibroblast-like cells (arrows) surrounding the regenerating nerve fibres in A. and form cellular sheet in the subperineurial space in B. PER, perineurium; ma, myelinated nerve fibres; s, Schwann cells, o, oedema; u, unmyelinated nerve fibers. 

DISCUSSION

Traumatic nerve injury and various pathological conditions result in the accumulation of endoneurial oedema; a salient feature in most types of neuropathies (Pedowitz et al.; Gabreëls-Festen et al.). Diabetes is one of the most common diseases that produce persistent peripheral neuritis as a result at least partially to the resulting endoneurial oedema secondary to effects on the vasa nervosa of the peripheral nerves (McManis et al.; Maxfield et al.).

Accumulation of endoneurial oedema is a result of damage to the blood-nerve barrier, which is consisted of the endoneurial blood vessels and the inner cellular layer of the perineurium. Consequently, the permeability of the endoneurial blood vessels increases and the accumulation of endoneurial oedema more in the subperineurial and perivascular spaces, although there is no clear explanation for such sites of accumulation (Pedowitz et al.). It results in an increase of intraneural pressure, which in turn reduces the blood flow in the endoneurium and endoneurial oxygen tension. The mechanical affection of the perineurial and epineurial blood vessels are observed to be resulted in degeneration of the nerve fibres (Low et al.; Gonda et al., 2010).

Ultra-structural studies of the recovering crushed peripheral nerves showed marked progressive reduction of the endoneurial oedema until it resolves completely (Pedowitz et al.; Gabreëls-Festen et al.). Consequently, nerve recovery after traumatic or pathological injury depends mainly on the resolution of endoneurial oedema, thus decreasing the intraneural pressure and increasing the endoneurial blood flow. It was reported that the maximum accumulation of endoneurial oedema was during the second week following the traumatic injury to the peripheral nerve, which coincides with the maximum increased permeability of the blood-nerve barrier (Pedowitz et al.). The rate of resolution of such an oedema will match the rate of functional nerve recovery. The fate of the endoneurial oedema, together with the cellular events and mechanisms of its resolution, remain unclear.

The present study showed that differentiated endoneurial fibroblast-like cells, in the segments distal to the site of the crush injury of the sural nerve, were apparently increased. These cells were flattened with long branching cytoplasmic processes that contained, in many cases, pinocytotic vesicles with areas of contacts in between. During the second week after nerve crush injury, the flattened fibroblast-like cells formed cellular barrier-like layers in the subperineurial space. During the third week following crush injury, a barrier-like cellular layer was a complete sheet of cells interrupted at sites where the endoneurial oedema communicated with the subperineurial oedema. The cellular sheet made by the fibroblast-like cells were isolated from the inner cellular layer of the perineurium by the accumulated subperineurial oedema which could explain the subperineurial accumulation of the endoneurial oedema following nerve injury.

The current study also showed that the fibroblastlike cellular barrier-like sheets probably aimed at supporting the disturbed inner cellular layer of the perineurium; one site of the blood-nerve barrier. Such subperineurial accumulation of oedema could also increase the fluid pressure in the subperineurial, which may help in the absorption of the endoneurial oedema through the perineurium into the epineurial blood vessels. The scattered endoneurial fibroblasts and the absence of fibroblast-like cellular barrier-like sheets in the normal nerves may support this hypothesis.

During the current study, the endoneurial fibroblastlike cells together with the collagen bundles wrapped around groups of the regenerated nerve fibres, thus creating endoneurial channel-like spaces in-between. These spaces were bounded on both sides by fibroblast-like cells and collagen bundles and contained degenerated myelin and endoneurial oedema. In most of the specimens, endoneurial macrophages made part of the wall of these channel-like spaces. The subperineurial space was in the communication of these spaces in certain areas of the endoneurium. Therefore, endoneurial oedema can reach the subperineurial where its absorption and resolution can occur. It is suggested that movements of the long processes of the fibroblast-like cells may create a current for the direction of the endoneurial oedema in these spaces towards the subperineurial. Moreover, isolation of the regenerated nerve fibres by fibroblast-like cells can help the regenerative process of the nerve fibers.

The stroma of the tumours contains an increased number of cancer-associated fibroblasts (CAFs) which are pro tumorigenic. There is growing evidence that CAFs are genetically different from the normal fibroblasts and could be potential targets for anti-cancer therapy.

Evidence that is epigenetically and possibly also genetically distinct from normal fibroblasts is beginning to define these cells as potential targets for anti-cancer therapy (Kobashigawa et al., 2011). On the other hand researchers found that the blood-borne fibroblast-like cells have the common surface markers of the hematopoietic stem cells CD34 (37).

Endoneurial Fibroblast-Like Cells might represent around 2 percent to 9 percent of the endoneurial cells. The spindle shaped cells on electron microscopy (EM) with rectangular or triangular bodies of cells when viewed in the transcerse section. They have long processes of cytoplasmic that expands either laterally between nerve fibers or along the nerve trunk; these types of processes usually loosely interdigitate with the neighbouring endoneurial fibroblast-like cells and also enclose one or various Schwann cells. In contrast with the Schwann cell nuclei, their nuclei seem paler and their cell membrane represents micropinocytotic vesicles in smooth invaginations. The cytoplasm of endoneurial fibroblast-like cells contains Golgi apparatus, prominent endoplasmic reticulum and scattered mitochondria which is frequently orientated and is dilated in places and more or less in parallel arrays. Granular material is present in the cisternae. These cells comprise of only a few lysosomes in normal nerves. It is very complex to distinguish between extremely convoluted cells and intracytoplasmic vacuolation observed in cross section. Intermittently, the cytoplasm of endoneurial fibroblast-like cells is invaginated by bundles of collagen that is present in the plasma membrane with direct contact (^{Richard et al., 2012}, 2014).

CONCLUSION

The results indicated that fibroblast-like cells might play an important role in localising the endoneurial oedema in the subperineurial space by forming barrier-like cellular sheets. These cells also lined channel-like endoneurial spaces that containeddegenerated myelin and oedema and are continuous with the sub-perineurial space. Consequently, fibroblast-like cells may play an important role in the localization of endoneurial oedema following crush injury. Immunohistochemistry study of the fibroblast-like cells in normal and crushed nerves is recommended.