The hemal node is a hemato-poietic and lymphoid organ found in some mammals such as ruminants, humans and rats. In ruminants, hemal nodes are located in the subcutaneous region of the head, mesenteric region and along the large blood vessels such as the aorta in the thorax and abdomen; however, the distribution and number of hemal nodes in these regions seem to be diverse (Casteleyn et al., 2008). The functions of hemal nodes had not been fully elucidated. Postulated functions included erythrophagocytosis, erythropoiesis, platelet formation, blood storage and filtration and immune functions in bovines (Cerutti and Guerrero 2008, Zidan et al., 2012). Keeping in view the diverse views expressed about the histophysiology of hemal nodes, the present study was planned to study distribution of various enzymes in hemal nodes of buffalo.

Materials and Methods

The present study was conducted on hemal nodes of buffalo (n=12) of different age groups. The fresh unfixed hemal nodes from buffalo were immediately collected after sacrifice and stored in liquid nitrogen. These tissues were subjected to cryostat sectioning at -20 ⁰ C with cryostat microtome. The sections of 10 -12 µm thickness were obtained on clean glass slides and were incubated for demonstration of various enzymes as alkaline phosphatase (AKPase), glucose-6-phosphatase (G-6-pase) by coupling azodye method (Barka and Anderson,1963), succinate dehydrogenase (SDH), lactate dehydrogenase (LDH), reduced nicotinamide adenine dinucleotide diaphorase (NADH-diaphorase), reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) and glucose-6-phosphate dehydrogenase (G-6-PD) by Nitro BT method (Pearse , 1972).

Result and Discussion                                          

The histoenzymic activity of different phosphatases and oxidoreductases of hemal nodes in buffalo is detailed below.

Phosphatases                                                      

Alkaline phosphatase (AKPase)

The alkaline phosphatase activity was constantly weak in the capsule of the hemal node. The subcapsular sinus showed weak (Fig. 1) to moderate activity whereas stroma of the hemal nodes showed weak activity for AKPase. The activity was moderate to strong in the lymphoid follicles at the periphery (Fig. 2), however weak activity was observed in the germinal centers of the lymphoid follicles (Fig. 3). Lymphoid follicles devoid of germinal centers showed moderate to strong activity. The diffused lymphocytes present loosely in the stroma /parenchyma of hemal nodes were moderately positive for alkaline phosphatase (Fig. 2). Similar observations were made by (Zidan and Pabst, 2004) in endothelium of the sinuses and lymphoid follicles. The frequent association of the enzyme alkaline phosphatase with monosaccharides in the tissue was observed and may be associated with the transport of nutrients (Kumar et al., 2017).

Glucose-6-Phosphatase (G-6-Pase)

Weak to mild activity for Glucose-6-phosphatase was observed in entire hemal node (Fig. 4). Glucose-6-phosphatase consisted of amino acids, anchored to the endoplasmic reticulum (ER) and is involved in the release of glucose into the circulation.

Oxidoreductases

Dehydrogenase

Succinic Dehydrogenase (SDH)

In the present study weak SDH activity was noted in the capsule of the hemal node whereas subcapsular sinus showed weak to moderate activity for SDH (Fig. 5). The stroma of the hemal node was weakly positive for SDH. SDH is a mitochondrial enzyme that is involved in generation of energy by oxidation-reduction reaction in the cell (Smith, 1969). This enzyme might be associated with oxidation of fatty acids that lead to formation of lipid pigment granules (Smith, 1969).

Glucose 6 Phosphate Dehydrogenase (G-6-PD)

In the present study, glucose 6 dehydrogenase activity was weak in the capsule and stroma. The cortex showed. Weak G-6-PD activity and lymphoid follicles showed weak activity (Fig. 6). The glucose 6 phosphate dehydrogenase enzyme activity is associated with the pentose phosphate shunt (Fennel and Pearse, 1961). These pentose phosphates might be utilized for nucleic acid synthesis during development. Turkoglu and Aldemir (2003) also reported the Glucose 6-phosphate dehydrogenase (G-6-PD) enzyme activity in all mammalian tissues especially in cytosol and mitochondria.

Lactate Dehydrogenase (LDH)

A moderate activity to lactate dehydrogenase was observed in the capsule of hemal node and lymphoid follicles. The subcapsular sinus showed weak to moderate activity for LDH. The diffused lymphocytes arranged loosely in the parenchyma showed weak to moderate LDH activity (Fig. 7). LDH is an NAD dependent enzyme found in cells in which glycolytic pathway is active. It catalyses the formation of lactate in anaerobic glycolysis and pyruvate in aerobic respiration. The presence of LDH enzyme in hemal nodes suggested the presence of glycolytic pathway in cellular elements especially in the capsule and lymphoid follicles.

Diaphorases

Reduced Nicotinamide Adenine Dinucleotide Diaphorase (NADH-D) and Reduced Nicotinamide Adenine Dinucleotide Phosphate Diaphorase (NADPH-D)

In the present study, weak nicotinamide adenine dinucleotide diaphorase was observed in the capsule of the hemal node (Fig. 8). The activity was weak to moderate in the stroma of the hemal node. In cortex, strong activity was noticed. The activity of this enzyme was intense in the lymphoid follicle (Fig .10 & 11). The periphery of the lymphoid follicle showed moderate activity to this enzyme (Fig. 9). The subcapsular sinus of hemal node showed weak enzyme activity. Connective tissue fibers showed moderate reaction to this enzyme. The cellular elements showed moderate to strong activity. However, intense activity was present in lymphoid follicles. Moderate activity to nicotinamide adenine dinucleotide phosphate diaphorase was present in the capsule of the hemal node (Fig. 9). The subcapsular sinus showed weak to moderate activity to NADPH. In stroma moderate activity of this enzyme was observed. The activity of this enzyme was strong to intense in lymphoid follicles. NADH and NADPH diaphorases are co-enzyme dehydrogenases and acts in the cell as a part of hydrogen transport chain. The enzyme intensity indicated metabolic activity of the cell.

Conclusion

The variable activity of phosphatases and oxidoreductases in hemal nodes of different age groups of buffalo reflected the development of metabolic pathways and maturation of cellular elements especially the lymphocytes.

References

1. Barka, T. and Anderson, P. J. (1963). Histochemistry: Theory, Practice and Bibliography, Published by Harper and Row Publishers, New York. pp 237-316.
2. Casteleyn, C., Breugelmans, S., Simoens, P., and Van den Broeck, W.( 2008). Morphological and immunological characteristics of the bovine temporal lymph node and hemal node. Veterinary Immunology and Immunopathology 126: 339-350.
3. Cerutti, P. and Guerrero, F. (2008). Erythropoiesis and erythrophagocytosis in bovine haemal nodes. International Journal of Morphology 26: 557-562.4. Fennel, R. A. and Pearse, A. G. E. (1961). Some histochemical observations on the bursa of fabricius and thymus of the chicken. Anatomical Record. 139:93-103.
4. Pearse, A, G, E. (1972). Histochemistry: Theoritical and Applied. 3^rd Vol II. Published by Churchill Livingstone, London.
5. Smith, C. (1969).Studies on the thymus of mammals XVI. Lipopigment cells in the cortex of the thymus of the mouse. American Journal of Anatomy 124:389-410.
6. Turkoglu V and Aldemir S. (2003). Purification and characterization of glucose 6-phosphate dehydrogenase from sheep liver. Turkish Journal of Chemistry 27: 395-402
7. Zidan, M. and Pabst, R. (2004). Histological, histochemical and immunohistochemical study of the haemal nodes of the dromedary camel. Anatomia Histologia Embryologia 33: 284–289.
8. Zidan, M., Zaghloul, D. M., Derbalah, A. E. and Elghoul, M. (2012). Age related morphological changes in haemal nodes of the Egyptian water buffalo (Bos bubalus). Alexandria Journal of Veterinary Science 37: 519-528
9. Kumar, V., Singh, S., Farooqui, M., Gangwar, C., Kumar, P., & Prakash, A. (2017). Acid and Alkaline Phosphatase Activity in Uterus of Goat in Different Stage of Pregnancy. International Journal of Livestock Research 7(7): 92-97. http://dx.doi.org/10.5455/ijlr.20170409100346