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15ULTRASTRUCTURAL PECULIARITIES OF PERINEURIAL CELLS OF CAPSULAR ELEMENTS OF DORSAL ROOT GANGLIA. ANIMAL MODEL OF STUDY.
Aliyarbayova A.,
Azerbaijan Medical University, Baku Senior teacher of department Cytology, Embryology and Histology Doctor of philosophy (PhD) in Medicine, Mehraliyeva G., Azerbaijan Medical University, Baku Senior teacher of department Cytology, Embryology and Histology
Doctor of philosophy (PhD) in Biology, Sadiqova G., Azerbaijan Medical University, Baku Senior teacher of department Cytology, Embryology and Histology Doctor of philosophy (PhD) in Medicine,
Nacafova T., Azerbaijan Medical University, Baku Senior teacher of department Cytology, Embryology and Histology
Mansimov A. Azerbaijan Medical University, Baku Senior teacher of department Cytology, Embryology and Histology
ABSTRACT
The ultrastructural morphology of perineurial cells had been examined in the capsular elements of dorsal root ganglia of rats. The material prepared for investigation taken from the proximal part of the spinal nerve at different levels of spinal cord. The object were dorsal root sensory ganglia of white rats. Investigation ultrastructural properties of perineurium is important because its maintains physical barrier as well as stable homeostatic condition in the peripheral nerve, also in dorsal root ganglia. The main composition of blood- nerve barrier formed by perineurial barrier, that protected nerve fibers (axons) from toxic substances. Under light and electron microscopy studies revealed the main cellular component of perineurium is perineurial cells and investigated its detailed ultrastructural peculiarities. We found that perineurial cells modulate their cytoplasmic structure to perform their barrier function. Morphological observations identified that perineurial cells contained a lot of number pinocytotic vesicles especially in face to face side of it and that shown participation of it in mediate active transcytotic transport.
Keywords: dorsal root ganglia, capsule, perineurial cells, ultrastructure, transmission electron microscopy.
Introductions. Spinal nerve roots (ventral and dorsal) emanated from spinal horns in spinal cord; by joining of roots formed peripheral nerves, comprising sensory, motor, autonomic nerve fibers and its covered by connective tissue layer that extension from the meningeal layer (dura mater, arachnoid, pia mater). On the way of dorsal roots situated accumulation of pseudounipolar neuronal cell bodies known as dorsal root ganglia. Axodendritic extension of these neurons covering with Schwan glial cells called nerve fibers, which divided into myelinated and unmyelinated [1, 2]. Dorsal root ganglia surrounded by capsule. Capsule composed of an outer collagenous epineurium and inner perineurium. Perineurium consisting of multiple concentric layers of specialized epithelioid myofibroblasts that surround the innermost endoneurium, which consists of myelinated and unmyelinated axons embedded in a looser meshwork of collagen fibers [3, 4]. Endo-neurial homeostasis is achieved by tight junction-forming endoneurial microvessels that control ion, solute, water, nutrient, macromolecule and leukocyte influx and efflux between the bloodstream and endoneurium, and the innermost layers of the perineurium that control interstitial fluid component flux between the freely permeable epineurium and endoneurium [5, 6].
Aim. The aim of this study was to investigate the morphological ultrastructural properties of perineurial
cells composing the inner layer capsule surrounding the dorsal root ganglia and relation its structure with its function.
Materials and methods. In this study, object of research was used dorsal root ganglia from 20 adult male rats with a weight of 180 - 200 grams. The animals were secured in a standard laboratory in pathogen-free condition. All procedures complied with the Principles of Laboratory and Animal Care established by the Azerbaijan Medical University.
The animals were deeply anaesthetized with keta-mine (Nembutal) and divided into 2 groups; from first groups animals the tissue blocks prepared by immersion method (without washing the vessels); the vessels of second groups animals were washed 1 minute at a pressure of 80 mm Hg with Hank's solution by catheter administered to the initial part of aortic arch; and perfused transcardially with a solution containing 2% formaldehyde, 2% glutaraldehyde and 0.1% picrin acid prepared in phosphate buffer (pH 7.4). The perfusion lasted 25 minutes at a pressure of 80 mm Hg. After in both groups animals a spinal canal was opened and dorsal root ganglions were removed from the soft tissue, then postfixed in 1% osmium acid solution during 2 hours in phosphate buffer. Afterwards specimens were washed in distilled water, dehydrated in alcohol, embedded in Spurr, Epon - Araldit medium and prepared
Results and discussions. The connective tissue layer (dura matter, arachnoidea, pia matter) surrounding the spinl cord extended to the periphery (peripheral spinal nerve), covered DRG and collectively called the capsule of the DRG. On the figure 1 demonstrated a part of DRG with its capsule under light (A) and electron microscope (B). As can be seen from the slide, that made by the immersion method, clearly visible accumulation of neurons in different size (small, medium, large) and different staining properties (lightly and densely); also seen surrounding the neurons a capsule of ganglion composing of densely stained, flattened structures, collagen fibers, which are sharply interlocking. Onhistological slide between neurons visible myelinated and unmyelinated nerve fibers, lumen of blood vessels with blood formed elements. On ultrastructural slide with arrowhead shown axonal hilllock that emanating from cell body of pseudounipolar neuron.
Figure 1. Microscopic (A) and ultrastructure (B) of capsule and nerve elements of the spinal sensory ganglion, that made by the immersion method. Explanation given in text. Semithin section (A), electronogramma (B). Stain: A -methylen blue, azur II and basic fuscin; B - uranil acetat and lead citrate. Magnification: A 20^m, B 10^m.
blocks according to general methods accepted in electron microscopy [7]. Obtained from blocks on ultratomes Leica EM UC7 semithin sections (thickness 1-2^m) were stained with methylene blue, azure II and basic fuchsine or with toluidine blue; after examination with Latimet (Leitz) microscope necessary parts of images were taken on Pixera (USA) digital camera.
In addition, gained silver and gold ultrathin sections from same blocks were stained with 2% uranyl acetate solution, then in 0.6% lead citrate made in NaOH 0.1N solution; each section was carefully examined under the 80-120 kV in Transmission Electron Microscope JEM-1400 (Jeol, Japan), photographed at different magnification and received the electron micrographs. This procedure continued until the block had been used.
In both light (fig. 1a) and under a small magnification on electron microscopes (fig. 1 b), it is difficult to determine the compatibility, composition or differences of the structural elements containing in the capsule (shown with red arrow on both slide), due to preparation of histological slide by immersion method.
The capsule of DRG composed of two layer: outer and inner. Outer layer known as epineurium, epineuria or epineurial layer that consists of mostly collagen fibers; inner layer composed of mesenchymal originating perineuria! cells (form perineurium) [1]. We revealed
on intravenous perfusion fixation preparations (fig.2), the outer layer - epineurium consisting of dense collagen fibers (mostly type I collagen), blood vessels (shown in both slide). In addition, in fig. 2b inside epi-neurium clearly visible lymph vessels and thin walled veins, individual fat cells and some elements of loose connective tissue. The inner layer of capsule appears under a light microscope as a thin strip of dark color (shown by the arrows 2 a, 2 b) because it is composed of extremely dense cells by name perineurial cells.
Figure 2. Semithin section obtained by intravascular perfusion of dorsal root ganglia of rat. The explanation given in text. Stain: A - methylene blue; B - azure II and basic fuchsine. Scale bar 50pm in both slide. Abbreviation: Epi-epineurium, Per-perineurium
The perineurium plays an important role in maintaining the integrity, stability of the internal peripheral nerve environment by creating a physical barrier, that's why the morphological investigation of perineurium (its cellular component - perineurial cell) is necessary.
Ultrastructural examination of the perineurium shows that it is composed of flattened cells, by name
perineurial cells, arranged in number 5-8 layers surrounded on both sides by a basal lamina (fig. 3a, b, c, d). It should be noted that the thickness of these cells reaches 950 nm only in the nuclear and paranuclear regions (fig 3a), but does not exceed 250 nm in the remaining thinnest peripheral parts of it (fig 3b, d).
Fig 2. Ultrastructure ofperineurial cells composing capsule of sensory spinal dorsal root ganglion. Content of central nucleus located region (A), paranuclear peripheral region (B, C, D). The explanation given in text. Stain: uranil acetat and lead citrate. Magnification: 500 nm in all electronograms. Abbreviation: PC - perineurial cell; NPC -nucleus of perineurial cell; CPC- cytoplasm of perineurial cell; BM-basal membrane.
This indicates that the perineurial cells belong to an extremely flattened cell type. In the paranuclear parts of perineurial cells are revealed mitochondria (shown by a single axis in fig. 3a), cisterns of granular endoplasmic reticulum and Golgi network (shown by a double arrow in fig. 3a), lysosomes, free ribosomes (fig. 3a). In some peripheral regions of perineurial cells observed relative thickness of the cytoplasm because of in these parts situated mitochondria with some special cristae (marked with a single axis in fig. 3 c). The branching thin cytoplasmic processes of this cells also coated by an external basal lamina (fig. 3b, 3d). The most characteristic peculiarities of the peripheral parts of these cells is the presence many number vesicles in contact with their luminal and abluminal surfaces (fig. 3d). That indicated participation of perineurial cells in active transcytotic transport. Occasionally, in addition to fused caveolas with plasma membrane, in the cytoplasm of the peripheral parts of perineurial cells are detected free caveolae, clathrin-coated pinocytotic bubbles, section of cisterns of granular endoplasmic reticulum, and free ribosomes. One of the most noticeable features of perineurial cells belonging capsule of the DRG is that they are possess many processes, except only for the inner layer facing to the endoneurial area.
Conclusion. These experience about ultrastructural properties of perineurial cells, studying in animal model, increase our knowledge and understanding about it function in maintaining the integrity of the internal peripheral nerve environment by creating a physical barrier. That barrier under different patological and physiologic condition, limits the entry of biologically active substances, infectious agents, toxins, some ions and blood-borne cells into the nerve bundles. Also helping to recommend a specific therapeutic application for chronic peripheral neuropathies and neuropathic pain.
This work is implemented basing on financial support of the Science Development Foundation under the President of the Republic of Azerbaijan. Qrant № EiF-2011-1(3)-82/44/3-M-6.
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