Structural Insights of the Glycogen Body in Domestic Chicken

There is a special unusual cellular mass in birds’ spinal cords known as glycogen body (corpus gelatinosum). Because of the specific topographical situation of this circumventricular organ, the structure of this organ on the cellular and subcellular level is of special interest with respect to the still unsolved functional problems aiming to find a relation between the structure and the function of this organ. Twenty domestic chickens were used in this study to demonstrate the structural peculiarities of this body from the histological point of view. Our results revealed that the glycogen body is a very delicate, small, ovoid circumventricular, transparent and gelatinous consistency structure embedded in the dorsal part of the lumbosacral region of spinal cord. The body has two cell zones; Peripheral one had regularly arranged large cells with metachromatic faint red cytoplasm and peripheral basophilic nuclei and central one with irregularly arranged cells that had different basophilic degrees, variable sizes, and peripheral nuclei. By transmission electron microscope, the cells of both zones showed deeply infolded electron dense peripherally located nuclei with prominent nucleoli that were rounded in the outer zone and ovoid in the central one. The cytoplasm was almost occupied with large dense masses of glycogen. No connective tissue was observed in the body except in the vicinity of the blood vessels that were more distributed in the center of the body than in its periphery. Conclusion: It could be concluded that the histophysiological relationship of the glycogen body still a mystery hoping that the incoming research can solve it.


Introduction
The domestic chicken vertebral column has a unique structure known as glycogen body occupying the dorsal rhomboid sinus and extends from the level of 26 to 29 spinal nerves [1,2]. This body is partly intrapial and partly subpial and its dorsal surface is covered by the spinal pia mater, from which a pial septum enters the interior of the organ, separating it into a larger dorsal part and a small ventral part. The central canal of the spinal cord passes through the ventral part of the glycogen body [3]. Histologically, the body classified into central and peripheral parts. The cells in the peripheral portion showing a more regular arrangement than the cells in the central portion [2]. The nature of these cells is distinctive as it composed from highly specialized and morphologically modified astroglial cells [3][4][5]. In general, these cells are polygonal with narrow cytoplasmic rim and peripheral nuclei [6]. The cytoplasm is fully laden with glycogen which has strong positive reaction to both Best's carmine stain and PAS reaction [3,4]. At the subcellular level, these cells have few short processes, dense irregular-shaped nuclei and fully loaded cytoplasm with glycogen. The cytoplasmic rim is rich in ribosomes, rough endoplasmic reticulum, and free heterogeneous lysosomes [7,8]. The blood supplies of the glycogen body ramify from a sinusoidal network which is better developed in the central portion than in the peripheral one [6]. The physiology of glycogen body is a controversial and very complicated issue and there is no clear-cut function till now, but many literature had suggested that the function of the glycogen body is mainly related to transmission of hydrostatic pressure changes during movements of the bird on the ground [9,10], distributing substances originating in the glycogen body to the CNS [11], playing a role in the metabolism of the neurons of the spinal cord [2,[11][12][13], and sharing in the processes of myelin formation in the central nervous system [14].

Material and Methods
Twenty white Leghorn chicken of 8-month-old of 2000-2200 gm were used for glycogen body in vitro assay. The birds were obtained from the faculty of agriculture, Zagazig University, Zagazig, Egypt. They were housed separately, one in each cage and fed synthetic ration and water ad libitum. They were kept in the laboratory for 2 days before euthanization. They were euthanized by injection of sodium Phenobarbital (100 mg/kg body weight) in the wing vein [15]. Spinal cord lumbosacral region segments were collected, some of them were fixed in Bouin's solution and others were fixed in 10% buffered neutral formalin for 48 hours. All Tissue samples were dehydrated in ascending grades of alcohols, cleared in xylene and embedded in paraffin then some segment sectioned in 5 microns and stained with H&E and PAS. Others samples were sectioned at 10 microns serial sections and stained with H&E to demonstrate the extension of the glycogen body and the cross-sectional area of it using a calibrating lens [16]. For E/M study, the samples were, fixed in 3% glutaraldehyde and processed for resin preparation; Glycogen body tissues washed in phosphate buffer, post-fixation in 2% osmium tetraoxide, Then washed again in the same buffer, dehydrated in ascending grades of alcohol followed by propylene oxide. Finally, infiltrated in a mixture of 50% propylene oxide and 50% resin then embedded in 100% resin. Semithin sections were obtained using Leica ultracut ultratome and stained with toluidine blue. Ultrathin sections were obtained from different areas after using semithin sections as a guide, then mounted and stained with uranyl acetate and lead citrate and studied in a Jeol 1010 TEM [17,18].

Gross appearance of the glycogen body
Dissection of the vertebral column revealed; delicate, ovoid circumventricular, transparent and gelatinous glycogen body which embedded in the dorsal part of the lumbosacral region of spinal cord at the level of L2, L3, L4, S1 and S2 segments ( Figure A1). It is separate structure and upon removal it leave widened dorsal fissure without any injury to the spinal cord tissue. Its cross-sectional area was maximal at L4. Rostral to L2 and caudal to S2 the body is much smaller, leaving a large space dorsally.

Light microscopic picture of the glycogen body
Histological examination of serial paraffin sections stained with H&E declared that glycogen body located dorsal to the spinal cord and divided into dorsal and ventral parts ( Figure A2). The ventral part appears enclosed the central canal ( Figure A3). It appeared highly vascularised particularly, in the central portion and no connective tissue were detected except in the vicinity of the blood vessels ( Figure  A4). Its cells are closely pressed polygonal cells with narrow cytoplasmic rim and pushed nuclei toward one edge of the cells ( Figure  A5). The cells appeared filled with PAS +ve material and there was zonal variation in the positive PAS reaction intensity ( Figure A6). Plastic section stained with toluidine blue revealed two cell zones; peripheral cells zone and central one The peripheral cells were large and regularly arranged with metachromatic faint red color with peripherally located basophilic nuclei. Most cells in this zone appeared occupied with granular eosinophilic material, but few cell clusters filled with homogenous translucent eosinophilic content. These cells were found most closely to the blood vessels ( Figure B1). The cells in the central zone were irregularly arranged, had different basophilic degrees, variable in sizes, and peripherally located nuclei. The cells basophilia was greater around the blood vessels. The cytoplasm was occupied with a basophilic granular material in most cells but, few cells contained dark homogenous basophilic circular bodies ( Figure B2). Most cells in the central part of the central zone had very light basophilic cytoplasm and others appeared evacuated with an intact cell membrane and peripheral nuclei ( Figure B3).

Transmission electron micrograph of the glycogen body
Transmission electron micrograph showed that the peripheral zone cells had a small amount of juxtanuclear cytoplasm and very thin cytoplasmic rim around the cell perimeter ( Figure B4). The central zone cells had more voluminous juxtanuclear cytoplasm and broader cytoplasmic rim around the cell perimeter ( Figure B5). Both zones cells showed electron dense nuclei that were deeply infolded and peripherally located toward one side of the cell with prominent nucleoli. The cytoplasm was almost occupied with large dense masses of glycogen which were free of the usual cell organelles with the exception of few; small clear membrane-limited vacuoles and membrane-bounded masses of glycogen. Most cellular organelles were present but almost located at the juxtanuclear regions between the glycogen masses and the nucleus; numerous mitochondria, well developed Golgi apparatus, round lysosomes, numerous ribosomes mainly polysomes and rough endoplasmic reticulum was widely distributed. Clear cell junctions appeared between adjacent cells at the cells perimeters that facing the voluminous cytoplasmic parts ( Figure  B6). Figure A1: Gross appearance of spinal cord L4 level glycogen body; delicate, ovoid circumventricular, transparent and gelatinous mass (black arrow), embedded in the dorsal part of the spinal cord lumbosacral region (arrow heads) and located inside the rhomboid sinus (white arrow). Figure A2: Glycogen body divided into dorsal (d) and ventral parts (v) and its ventral part appear enclosed the central anal (arrow) (H&E.X4). Figure A3: Spinal cord L4 level glycogen body showing the central canal (arrow) that completely enclosed with the glycogen body cells (arrowheads) (H&E.X40). Figure A4: Spinal cord L3 level glycogen body appeared with high vascularisation (arrows) (H&E.X 10). Figure A5: Spinal cord L3 level glycogen body polygonal cells (arrows), empty cytoplasm (C) and pushed nuclei toward one edge of the cells (arrowheads) (H&E X.40). Figure A6: Spinal cord L2 level glycogen body cells appeared filled with PAS +ve material (arrowheads) (PAS stain X.20).  Figure B3: Spinal cord L4 level glycogen body plastic section central zone most inner cells that had very light basophilic cytoplasm (f) and some appeared evacuated with intact cell membrane and peripheral nuclei (c) (Toluidine blue X40). Figure B4: Transmission electron micrograph showing the peripheral zone cells with small amount of juxtanuclear cytoplasm (arrow) and very thin cytoplasmic rim around the cell perimeter (arrowheads) and most of the cell filled with glycogen particles (g) (E/M X5000). Figure B5: Transmission electron micrograph showing the central zone cells with voluminous juxtanuclear cytoplasm (arrow) and broader cytoplasmic rim around the cell perimeter (arrowheads) and perinuclear circular membranous structure (s) (E/M X4000). Figure B6: Transmission electron micrograph showing the peripheral zone cells with electron dense nucleus (N), numerous polysomes clusters (arrowhead), mitochondria (arrow) rough endoplasmic reticulum (r) and cell junctions (j) (E/M X3000).

Discussion
Although some litterateurs reported that the glycogen body extended along the whole length of the spinal cord [2], this study was designed to investigate the glycogen body in the lumbosacral region only because the typical circumscribed gelatinous mass present only in this region [13,19]. Upon dissection, the vertebral column glycogen body tissue was completely separate and easily removed from the nervous tissue of the spinal cord. This indicates that there is no interdigitation between the cord and the body [3]. Based up on the present findings; the area of the vertebral canal increases from L2 to reach its maximum at L4 with a subsequent decrease toward more caudal segments. This enlargement in the vertebral canal is not due to an increase in the size of the spinal nervous tissue but to the large glycogen body embedded in the dorsal rhomboid sinus [20]. The best method for soft tissue description and proper locations determination of the glycogen body was done by using razor blade sections because fixation in formalin cause soft tissue (spinal cord and glycogen body) shrinkage [21]. This is confirmed by the presence of a free space between the spinal cord and the glycogen body in paraffin sections and between dorsal and ventral part of the body. Although the glycogen body cells are of glial origin properly astrocytes [5] yet its morphology is completely different as they undergo extreme differentiation [22]. They are compressed polygonal cells of variable sizes, with narrow cytoplasmic rim and the nuclei pushed toward one edge of the cells. These cells are filled with PAS +ve material. The nature of this material has been approved histochemically, and by electrophoreses that it is glycogen [3,4,23,24]. According to previous reports and the present results the glycogen body cells could be classified morphologically into peripheral cell zone and central one [6,21]. The cells in peripheral zone are regularly arranged, large sized with peripheral basophilic nuclei and metachromatic faint red color with toluidine blue. This metachromasia might be due to interaction between carbohydrate macromolecules and toluidine blue [16,24]. The cells in the central zone are irregularly arranged, have variable sizes with peripheral nuclei. Its cytoplasm has different basophilic degrees with toulidine blue [2]. The glycogen accumulation in glycogen body cells may act as a possible source of energy for the avian central nervous system [25,26]. Many reports hypothesized that the transition in glycogen contents indicate that this body might be metabolically active and play a role in the metabolism of the neurons in the chicken spinal cord [2,12,13]. Other reports mentioned that these cells may be metabolically inert and their function related to the processes of myelin formation in the avian central nervous system in which such glycogen may serve as a source of organic acids which might provide alternate substrates to the CNS under conditions of metabolic stress [14]. In contrary few reports hypothesized that this organ involved in the equilibrium of locomotion and transmission of hydrostatic pressure changes during movement on the ground [8,19].