Immunohistochemical Staining for Nerve Fibers in Normal Tissues with Frozen Sections: Structure and Function Rela-tionship
Main Article Content
Abstract
Small nerve fibers are difficult to immunostain with routinely formalin-fixed and paraffin-embedded sections. Normal tissues except central nervous system are not densely innervated compared to densely distributed lymphatic and blood vessels and nerve fibers are not properly preserved in the routinely formalin-fixed and paraffin-embedded tissues. Nerve density technique with skin punch biopsy is an established method using 50 µm tissue sections, which are initially preserved in fixative. The fixed tissues are frozen sectioned and floating tissue sections are immunostained for nerve fiber markers. We had developed an alternative method using unfixed frozen sections mounted on glass slides. We had previously used this frozen section method for lymphatic and blood vessel immunohistochemistry and extended to nerve fiber immunostaining using neurofilament and CD56 as nerve fiber markers. Immunostained tissues with frozen sections were compared to formalin-fixed and paraffin-embedded colon and kidney sections. The normal tissues from rhesus monkey included heart, intestines, diaphragm, pancreas, spleen, kidney, thyroid, urinary bladder and others. Frozen section immunostained sections clearly depicted much more fine nerve fibers especially using CD56 as a nerve marker than with paraffin-embedded tissues. This technique is more labor-intensive but provides more precise distribution of fine nerve fibers than with routinely paraffin-embedded tissues. This immunostaining with frozen sections validated usefulness to depict more detailed nerve distribution and will shed light on normal histology and histopathology.
Keywords:
CD56, frozen sections, immunohistochemistry, nerve fibers, neurofilament, normal rhesus monkey tissues
Article Details
How to Cite
TOMITA, Tatsuo.
Immunohistochemical Staining for Nerve Fibers in Normal Tissues with Frozen Sections: Structure and Function Rela-tionship.
Medical Research Archives, [S.l.], v. 11, n. 6, july 2023.
ISSN 2375-1924.
Available at: <https://esmed.org/MRA/mra/article/view/3884>. Date accessed: 15 may 2024.
doi: https://doi.org/10.18103/mra.v11i6.3884.
Section
Research Articles
The Medical Research Archives grants authors the right to publish and reproduce the unrevised contribution in whole or in part at any time and in any form for any scholarly non-commercial purpose with the condition that all publications of the contribution include a full citation to the journal as published by the Medical Research Archives.
References
1. Kerr, JB: Nervous system. In : Kerr, JB, Atlas of Functional Histology, ed. JB Kerr, Mosby, Lon-don, 1998; 107-110.
2. Litchfield, S, Nagy, Z: New treatment modifi-cation makes the Bielschowsky silver stain re-producible. Acta Neuropath 2001(1);17-21.
3. Uchihara, T: Silver diagnosis in neuropathies: principles, practice and revised interpretation. Acta Neuropath 2007, 113(5);483-499.
4. Tokushige, N, Marham, R, Russell, P, Fraser, IS: High density of small nerve fibers in the func-tional layer of the endometrium in women with endometriosis. Hum Reprod 2006, 21(3);782-787.
5. Tokushige, N, Markham, R, Russell, P, Fraser, IS: Different types of small nerve fibers in eu-topic endometrium and myometrium in women with endometriosis. Fert Steril 2007, 88(4);795-803.
6. Boker, A, Kyama, CM, Vercruysse, L, Fass-bender, Gevaert, O et al: Density of small di-ameter sensory nerve fibers in endometrium: a semi-invasive diagnostic test for minimal to mild endometriosis. Human Reprod 2009, 24(12);3025-3032.
7. Tokushige, N, Markham, R, Russell, P, Fraser, IS: Effects of hormonal treatment in nerve fi-bers in endometrium and myometrium in wom-en with endometriosis. Fertil Steril 90(5);1589-1598.
8. McArthur, JC, Stocks, EA, Hauer, P, Cornblath, DR, Griffin, JW: Epidermal nerve fibers densi-ty. Arch Neurol 1998, 55(12);1513-1520.
9. Ebenzer, GS, Hauer, P, Gibbons, C, McArthur, JC, Polydefkis, M: Assessment of epidermal fi-bers: A new diagnostic and predictive for pe-ripheral neuropathies. J Neuropath Exp Nerol 2007, 66(12);1059-1073.
10. Saperstein, DS, Leveine, TD, Hank, N: Useful-ness of skin biopsy in the evaluation and man-agement of patients with suspected small nerve neuropathy fiber neuropathy. Int J Neu-rosci 2013,123(1);38-41.
11. Grayston, R, Czanner, E, Elhadd, K, Goebel, A, Frank, B et al: A systematic review and meta-analysis of the prevalence of small fiber in pa-thology in fibromyalgia of a new paradigm in pathology in fibromyalgia etiogenesis. Sem Arthritis Rheumat 2019, 48(5);933-949.
12. Tomita, T, Mah, K: Cyclic changes of lymphatic and venous vessels in human endometrium. Open J Pathol 2014, 4(1);5-12.
13. Tomita, T, Mah, K: Cyclic changes of nerve fibers in human endometrium. Open J Pathol 2014, 4(1);68-78.
14. Tomita, T: Immunohistochemical staining for lymphatic and blood vessels in normal tissues: comparison between routinely paraffin-embedded tissues and frozen sections. Acta Medica Acad 2120, 50(1);13-28.
15. Thompson, RS, Doran, JF, Jackson, P, Dhillon, AP, Rode, J: PGP9.5 -A new marker for verte-brate neurons and neuroendocrine cells. Brain Res 1983, 278(1-2);224-228.
16. Wilkinson, KD, Lee, KM, Deschpande, S, Deschpande-Hughs, P, Boss, JM: The neuron specific protein PGP9.5 is ubiquitin carboxy-terminal hydroxylase . Science 1989, 246(4930);670-673.
17. Karanth, SS, Springall, DR, Kuhn, DM, Levine, MM, Pollak, JM: An immunocytochemical study of neuropeptides and protein gene 9.5 in man and common employed laboratory animals. Am J Anat, 1991, 191(4);369-383.
18. Schlepfer, WW: Neurofilaments: structure, metabolism and implications in disease. J Neu-ropathol Exp Neurol 1987, 46(2):117-129.
19. Yuan, A, Rao, MV, Veeranna, Nixon, RA: Neu-rofilaments at a glance. J Cell Sci 2012, 125(14);3257-3263.
20. Hall, J: Guyton and Hall Textbook of Medical Physiology, 12th ed., Saunders/Elsevier, Phila-delphia, PA, 2011, 563-564.
21. Nagi, SS, Marshall, AG, Makdani, MA, Jaro-cha, E, Lifjencranz, J et al: An ultrafast system for signaling mechanical pain in human skin. Sci Adv 2019, 5(7);1297.
22. Mechtersheimer, G, Stauder, M, Moller, P : Expression of the natural killer cell--associated antigen CD56 and CD57 in human neural and striated muscle cells and their tumors. Cancer Res 1991, 51(5);1300-1307.
23. Jensen,M, Berthold, F: Targeting the neural cell adhesion in molecules in cancer. Cancer Lett 2007, 258(1);9-21.
24. Enriquez-Barreto, L, Palazzetti, C, Brenonann, LH, Maness, PF, Fairen, A: Neural cell adhesion in molecule, NCAM, regulates thalamocortical axon pathfinding and the organization of cor-tical somatosensory regeneration in mouse. Fron Molec Neurosci 2012, 00076.
25. Leshcynska, I, Liew, HT, Shephard, C, Halliday, GM, Stevens, CH: Aβ-derived reduction of MCA M2 -mediated synaptic adhesion con-tributes to synaptic loss in Alzheimer’s disease. Nature Common 2015, 6;8836.
26. Lajoix, AD, Reggio, H, Chardes, T, Peraldi-Roux, S, Tribillac, E et al: A neural isoform of nitric oxide synthase expression in pancreatic β-cells control insulin secretion. Diabetes 2001, 5(6);1311-1323.
27. Bahadoran, Z, Caristyrom, M, Mirmiran, P, Ghasemi, A: Nitric oxide: To be not to be en-docrine hormone? Acta Physiolo 2020,229(1);e13443.
28. Wheeler, T, Watkins, PS: Cardiac denervation in diabetes. Br J Med 1973, 4;584-586.
29. Marron, K, Wharton, J, Shepard, MN, Fagan, D, Royston, D et al: Distribution, morphology, and neurochemistry of endocardial and epi-cardial nerve terminal arborization in the hu-man heart. Circulation 1995, 92(8);2343-2351.
30. Armour, JA, Murphy, DA, Yuan, BX, MacDon-ald, S, Hopkins, DA: Gross and microscopic anatomy of the human intrinsic cardiac nerv-ous system. Anat Rec 1999, 247(2);289-298.
31. Michell, GA: Innervation of the heart. Br Heart J, 1953,15(2);159-171.
32. Cavallotti, C, Bruzzone, P, Mancone, M: Cate-cholaminergic nerve fibers and beta-adrenergic receptors in the human heart and coronary vessels. Heart Valves 2002, 17(1);30-35.
33. Hanna, P, Rajendran, PS, Ajiola, O, Vaseghi, M, Andrew, J et al: Cardiac neuroanatomy-imaging nerves to define functional control. Auton Neurosci 2017,207;48-58.
34. Christensen, AH, Nygaard, S, Rolid, K, Ny-troen, K, Gullestad, L et al: Early signs of sino-atrial reinnervation in the transplanted heart. Transplantation 2021, 105(9);2086-2096.
35. Grupper, E, Gewirtz, H, Kushwaha, S: Reinner-vation post-transplantation. Eur J Heart, 2018, 39(20);1799-1806.
36. Crick, SJ, Wharlton, J, Sheppard, MN, Royton, D, Yacoub, MH et al: Innervation of the human cardiac conduction system: A quantitative im-munohistochemical and histochemical study. Circulation 1994, 89(4);1697-1708.
37. Sparrow, MP, Weichselbaum, M, McCry, B Jr: Development of the innervation and airway smooth muscle in human fetal lung. Am J Respir Cell Mol Biol 1999, 20(4);550-560.
38. Lauwryns, JM, Van Ranst, L: Protein gene product 9.5 expression in the lungs of humans and other mammals. Immunocytochemical de-tection in neuroepithelial bodies, neuroendo-crine cells and nerves. Neurosci 1988, 85(3);311-316.
39. Laitinen, A: Autonomic innervation of the hu-man respiratory tract as revealed by histo-chemical and ultrastructural method. Eur J Respir Dis 1985, 140(Suppl);1-42.
40. Fisher, A, Hoffmann, B: Nitric oxide synthase in neurons and nerve fibers of lower airways and in vagal sensory ganglia of man. Correlations with neuropeptides. Am J Respir Crit Med 1996, 154(1);209-216.
41. Hiayet, MA, Wiid, HA, Wilson, AS: Investiga-tions on the innervation of the human dia-phragm. Anat Rec 1974, 179(4);507-516.
42. Martin, M, Li, K, Wright, MC, Lepore, AC: Functional and morphological assessment of diaphragm innervation by phrenic motor neu-rons. J Vis Exp 2015, 99;e52605.
43. Verlinden, TJM, van Djik, P, Herrier, A, de Gierde Vries, C, Lamers WH et al: The human phrenic nerve serves as morphological conduit for autonomic nerves and innervates the caval body of the diaphragm. Scientific Rep, 2018, 8;11697.
44. Young, RL, Page, AJ, Cooper, NL, Fishby, CL, Blickshow, LA: Sensory and motor innervation of the crural diaphragm by the vagal nerve. Gastroenterol 2010, 138(3);1091-1101.
45. Gazami, BA, Srinivassan, S: Enteric nerve sys-tem in the small intestine: Pathophysiology and clinical implication. Curr Gastroenter Rep 2010, 12(5);358-365.
46. Garcia-Victoria, M, Garcia-Corchon, C, Ro-drigues, JA, Garcian-Amigot, E, Burrel, MA: Expression of neuronal nitric oxide synthase in several type of the rat gastric epithelium. J Histochem Cytochem 2000, 48(8);1111-1119.
47. Timermans, JP, Babiers, M, Scheurmann, DW, Bogers, JJ, Adriansen, D et al: Nitric oxide synthase immunoreactivity in the enteric nerv-ous system of the developing human digestive tract. Cell Tissue Res 1994, 275(2);235-245.
48. Wattchow, DA, Brooks, SJ, Cost, M: The mor-phology and projections of retrogradely la-belled myenteric neurons in the human intes-tines. Gastroenterol 1995, 109(3);866-875.
49. Porter, AJ, Wittchow, DA, Brooks, SIH, Schur-mann, M, Costa, M: Choline acetyltransferase immunoreactivity in the human small and large intestine. Gastroenterol 1996, 111(2)401-408.
50. Assouz, LL, Sharma, S: Physiology, Large intes-tine. Stat Pearls, August 9, 2021.
51. Felten, DL, Ackerman, KD, Wiegand, SJ, Fel-ten, SY: Noradrenergic sympathetic innerva-tion of the spleen. 1. Nerve fibers associate with lymphocytes and macrophages in specific compartments of the splenic white pulp. J Neu-rosci Res 1987,181(1);28-36.
52. Bujis, R, van der Villet, J, Garidodus, ML, Huntinga, I, Escobar, C: Spleen vagal dener-vation inhibits the production of antibodies to circulating antigens. PLOS 3(9);e3152,2008.
53. Hu, D, Al-Shalan, MHA, Shi, Z, Wang, P, Wu, Y et al: Distribution of nerve fibers and nerve-immune cell association in mouse spleen re-vealed by immunofluorescent staining. Sci Rep 10;9850, 2020.
54. Giogrio, R, Sternini, C, Anderson, K, Brecha, N, Go, V: Tissue distribution and innervation pat-tern of peptide immunoreactivity in the rat pancreas. Peptides 1992, 13(1);91-98.
55. Ushiki, T, Watanabe, S: Distribution and ultra-structure of the autonomic verves in the mouse pancreas. Microscop Res Tech 1997, 37(5-6);399-406.
56. Rodriguez-Diaz, R, Caicedo, A: Novel ap-proach to studying the role of innervation plays in the biology of pancreatic islets. Endo-crinol Metab Clin North Am 2013, 42(1);39-56.
57. Chandra, R, Liddle, RA: Neural and hormonal regulation of pancreatic secretion. Curr Oppin Gastroenterol 2014, 30(5);490-494.
58. Tomita, T, Lacy, PE, Matschinsky, FM, McDan-iel, M: Effects of alloxan on insulin secretion I isolated rat islets perifused in vitro. Diabetes 1974, 23(6);517-524.
59. Barthout, HR, Powley, TL: Identification of va-gal preganglion that mediates cephalic phase insulin. Am J Physiol 1990, 258(2);R523-R530.
60. Li, W, Yu, G, Liu, Y, Sha, L: Intrapancreatic ganglia and neural regulation of pancreatic endocrine secretion. Front Neurosci 13;1-10,2019.
61. Ahren, B, Taborsky, GJJr: The mechanism of vagal nerve stimulation of glucagon and insu-lin secretion. Endocrinol 1986, 118(1);1551-1557.
62. Tomita, T, Friesen, SR, Kimmel, JR, Pollock, HG: Pancreatic polypeptide-secreting islet cell tu-mors. Am J Pathol 1983, 113 (2);134-142.
63. Mudler, J, Hokfelt, T, Knuepfer, MM, Kopp, UC: Renal sensory and sympathetic nerves re- innervate kidney in a similar time-dependent fashion after renal denervation in rats. Am J Physiol Rregul Integr Physiol 2013, 304(8);R675-R682.
64. Barajas, L, Powers, K: Monoaminergic innerva-tion of rat kidney. A quantitative study. Am J Physio 259(pt2);F503-F511.
65. Mitchell, AGE: The nerve supply of the kid-neys. Acta Anat 1950, 10(1);1-37.
66. Di Bona, GF, Kopp, UC: Nerve control of renal function. Phys Rev 1997, 77(1);75-197.
67. Ahren, B: Thyroid neuroendocrinology: Nerve regulation of thyroid hormone. Endocr Rev 1986, 7(2);149-155.
68. Amenta, F, Caporuscio, D, Ferrante, F, Porcelli, F, Zomparelli, M: Cholinergic nerves in the thy-roid gland. Cell Tissue Res 1978, 195(2);367-370.
69. Grunditz, t, Hakenson, R, Rerup, C, Sundler, F, Uddman, R: Neuropeptide Y in the thyroid gland: neuronal localization and enhancement of stimulated thyroid hormone secretion. Endo-crinol 115(4);1537-1542.
70. Silva, JE, Bianco, SD: Thyroid adrenergic in-teractions: Physiological and clinical implica-tions. Thyroid 2008, 18(2);157-165.
71. Parker, TL, Kesse, WK, Mohamed, AA, Afe-work, M: The innervation of the mammalian adrenal gland. J Anat 1993, 183(pt 1);265-276.
72. Charlton, BG, Nkomazana, OF, Mc Gadey A, Neal, DE: A preliminary study of acetylcho-line-esterase-positive innervation in the human adrenal cortex. J Anat 1991, 176;99-104.
73. Holgert, H, Gagerlind, A, Hokfelt, T: Immuno-histochemical characterization of epidermic innervation of the rat adrenal. Horm Metab Res 1998, 30(6-7);3156-3222.
74. Murabayashi, H, Kuramoto, H, Kawano, H, Sasaki, M, Katamura, N et al Immunohisto-chemical feature of substance p-immunoreactive chromaffin-cells and nerve fi-bers in the rat adrenal gland. Arch Histol Cytol 2007, 70(3);183-196.
75. Heym, C, Braun, B, Shuji, Y, Klimasschewski, L, Colombo-Benkmann, M: Immunohistochemical correlation of human adrenal verve fibers and thoracic dorsal root neurons with special ref-erence to substance P. Histochem Cell Biol 1995, 104(3);233-243.
76. Fung, MM, Miveerros, OH, O’Conner, DT: Dis-eases of the adrenal medulla. Acta Physiol 2007, 192(2); 325-335.
77. Ji, RC, Kato, S: Intrinsic interrelationship of lymphatic endothelia with nerve elements in the monkey urinary bladder. Acta Rec 2000, 259(1);86-96.
78. Yoshimura, N, Chancellor, MB: Neurophysiolo-gy of the lower urinary tract function and dys-function. Rev Urol 2003, 5(Suppl 8);s3-s10.
79. de Groat, W: Integrative control of the lower urinary tract: preclinical perspective. Br J Pharmacol 2006, 147(Suppl 2);s25-s40.
80. Andersson, KE: Bladder activation: afferent mechanisms. Urol 2002, 39(Suppl 1);43-50.
81. Bauer, MM, Smith, PG: Estrogen and female reproductive tract innervation: Cellular and molecular mechanisms of autonomic neuroplas-ticity. Auton Neurosci 2015, 187(1);1-17.
82. Hoyle, CV, Stones, RW, Robson, T, Whitley, K, Burnstock, GY: Innervation of vascular and mi-crovasculature of the human vagina by NOS and neuropeptide-containing nerves. J Anat 1996, 188(3);633-644.
83. Li, T, Liau, H, Gao, X, Li, X et al: Anatomic dis-tribution of nerves and microvascular density in the human anterior vaginal wall: Prospec-tive study. PLOS One, 2014, 9(11):e110239.
84. Griebling, TL, Liao, Z, Smith, PG: Systemic and tropical hormone therapies reduce vaginal in-nervation density in postmenopausal women. Menopause 2012, 19(6);630-635.
85. Bae, SE, Corcoran, BM, Watson, DE: Immuno-histochemical study of the distribution of ad-renergic and peptidermic innervation in the equine uterus and the cervix. Reproduction 2001, 122 (2);275-282.
86. Spezio Sardo, D, Floria, A, Soss Fernandez, LM, Guerra, G, Spinelli, M et al: The potential role of endometrial nerve fibers in the patho-genesis of pain generation during endometrial biopsy at office hysteroscopy. Reprod Sci 2015, 22(1);124-131.
87. Donadio, V, Nolano, M, Porovitera, V, Stancanelli, A, Lullo, F et al: Skin sympathetic adrenergic innervation: an immunofluores-cence confocal study. Am Neurol 2006, 59(2);376-381.
88. Lauria, G, Deviglia, G: Skin biopsy as a diag-nostic tool in peripheral neuropathy. Nat Clin Prac 2007, 3(10);546-557.
89. Wang, N, Gibbons, CH, Freeman, R: Novel immunohistochemical technique using discrete signal amplification systems for human cutane-ous peripheral nerve fiber imaging. J Histo-chem Cytochem 2011, 59(4);382-390.
90. Iwami, D, Ohi, R, Nio, M, Shimaoka, S, Sano, N: Abnormal distribution of nerve fibers in the liver of biliary atresia. Tohoku J Exp Med 1997, 181(1);57-65.
91. Akiyoshi, H, Gonad, T, Terada, T: A compara-tive histochemical and immunohistochemical study of aminergic, Cholinergic and peptider-mic innervation in rat, hamster, guinea pig, dog and human liver. Liver 1998, 18(5);352-359.
92. Grant, WF, Nicol, L, Thorn, SR, Grove, KL, Friedman, JR et al: Peripheral exposure to a high-fat diet is associated with reduced hepat-ic sympathetic innervation in one-year male Japanese macaques. PLOS One 2012, 7(10):e48119.
93. Tomita, T: Localization of nerve fibers in colon-ic polyp, adenoma and adenocarcinoma by immunocytochemical staining for PGP 9.5. Dig Dis Sci 2012, 57(2);364-370.
94. Tomita, T: PGP 9.5 immunocytochemical stain-ing for pancreatic endocrine tumors. Islets 2013, 5(1);1-7.
2. Litchfield, S, Nagy, Z: New treatment modifi-cation makes the Bielschowsky silver stain re-producible. Acta Neuropath 2001(1);17-21.
3. Uchihara, T: Silver diagnosis in neuropathies: principles, practice and revised interpretation. Acta Neuropath 2007, 113(5);483-499.
4. Tokushige, N, Marham, R, Russell, P, Fraser, IS: High density of small nerve fibers in the func-tional layer of the endometrium in women with endometriosis. Hum Reprod 2006, 21(3);782-787.
5. Tokushige, N, Markham, R, Russell, P, Fraser, IS: Different types of small nerve fibers in eu-topic endometrium and myometrium in women with endometriosis. Fert Steril 2007, 88(4);795-803.
6. Boker, A, Kyama, CM, Vercruysse, L, Fass-bender, Gevaert, O et al: Density of small di-ameter sensory nerve fibers in endometrium: a semi-invasive diagnostic test for minimal to mild endometriosis. Human Reprod 2009, 24(12);3025-3032.
7. Tokushige, N, Markham, R, Russell, P, Fraser, IS: Effects of hormonal treatment in nerve fi-bers in endometrium and myometrium in wom-en with endometriosis. Fertil Steril 90(5);1589-1598.
8. McArthur, JC, Stocks, EA, Hauer, P, Cornblath, DR, Griffin, JW: Epidermal nerve fibers densi-ty. Arch Neurol 1998, 55(12);1513-1520.
9. Ebenzer, GS, Hauer, P, Gibbons, C, McArthur, JC, Polydefkis, M: Assessment of epidermal fi-bers: A new diagnostic and predictive for pe-ripheral neuropathies. J Neuropath Exp Nerol 2007, 66(12);1059-1073.
10. Saperstein, DS, Leveine, TD, Hank, N: Useful-ness of skin biopsy in the evaluation and man-agement of patients with suspected small nerve neuropathy fiber neuropathy. Int J Neu-rosci 2013,123(1);38-41.
11. Grayston, R, Czanner, E, Elhadd, K, Goebel, A, Frank, B et al: A systematic review and meta-analysis of the prevalence of small fiber in pa-thology in fibromyalgia of a new paradigm in pathology in fibromyalgia etiogenesis. Sem Arthritis Rheumat 2019, 48(5);933-949.
12. Tomita, T, Mah, K: Cyclic changes of lymphatic and venous vessels in human endometrium. Open J Pathol 2014, 4(1);5-12.
13. Tomita, T, Mah, K: Cyclic changes of nerve fibers in human endometrium. Open J Pathol 2014, 4(1);68-78.
14. Tomita, T: Immunohistochemical staining for lymphatic and blood vessels in normal tissues: comparison between routinely paraffin-embedded tissues and frozen sections. Acta Medica Acad 2120, 50(1);13-28.
15. Thompson, RS, Doran, JF, Jackson, P, Dhillon, AP, Rode, J: PGP9.5 -A new marker for verte-brate neurons and neuroendocrine cells. Brain Res 1983, 278(1-2);224-228.
16. Wilkinson, KD, Lee, KM, Deschpande, S, Deschpande-Hughs, P, Boss, JM: The neuron specific protein PGP9.5 is ubiquitin carboxy-terminal hydroxylase . Science 1989, 246(4930);670-673.
17. Karanth, SS, Springall, DR, Kuhn, DM, Levine, MM, Pollak, JM: An immunocytochemical study of neuropeptides and protein gene 9.5 in man and common employed laboratory animals. Am J Anat, 1991, 191(4);369-383.
18. Schlepfer, WW: Neurofilaments: structure, metabolism and implications in disease. J Neu-ropathol Exp Neurol 1987, 46(2):117-129.
19. Yuan, A, Rao, MV, Veeranna, Nixon, RA: Neu-rofilaments at a glance. J Cell Sci 2012, 125(14);3257-3263.
20. Hall, J: Guyton and Hall Textbook of Medical Physiology, 12th ed., Saunders/Elsevier, Phila-delphia, PA, 2011, 563-564.
21. Nagi, SS, Marshall, AG, Makdani, MA, Jaro-cha, E, Lifjencranz, J et al: An ultrafast system for signaling mechanical pain in human skin. Sci Adv 2019, 5(7);1297.
22. Mechtersheimer, G, Stauder, M, Moller, P : Expression of the natural killer cell--associated antigen CD56 and CD57 in human neural and striated muscle cells and their tumors. Cancer Res 1991, 51(5);1300-1307.
23. Jensen,M, Berthold, F: Targeting the neural cell adhesion in molecules in cancer. Cancer Lett 2007, 258(1);9-21.
24. Enriquez-Barreto, L, Palazzetti, C, Brenonann, LH, Maness, PF, Fairen, A: Neural cell adhesion in molecule, NCAM, regulates thalamocortical axon pathfinding and the organization of cor-tical somatosensory regeneration in mouse. Fron Molec Neurosci 2012, 00076.
25. Leshcynska, I, Liew, HT, Shephard, C, Halliday, GM, Stevens, CH: Aβ-derived reduction of MCA M2 -mediated synaptic adhesion con-tributes to synaptic loss in Alzheimer’s disease. Nature Common 2015, 6;8836.
26. Lajoix, AD, Reggio, H, Chardes, T, Peraldi-Roux, S, Tribillac, E et al: A neural isoform of nitric oxide synthase expression in pancreatic β-cells control insulin secretion. Diabetes 2001, 5(6);1311-1323.
27. Bahadoran, Z, Caristyrom, M, Mirmiran, P, Ghasemi, A: Nitric oxide: To be not to be en-docrine hormone? Acta Physiolo 2020,229(1);e13443.
28. Wheeler, T, Watkins, PS: Cardiac denervation in diabetes. Br J Med 1973, 4;584-586.
29. Marron, K, Wharton, J, Shepard, MN, Fagan, D, Royston, D et al: Distribution, morphology, and neurochemistry of endocardial and epi-cardial nerve terminal arborization in the hu-man heart. Circulation 1995, 92(8);2343-2351.
30. Armour, JA, Murphy, DA, Yuan, BX, MacDon-ald, S, Hopkins, DA: Gross and microscopic anatomy of the human intrinsic cardiac nerv-ous system. Anat Rec 1999, 247(2);289-298.
31. Michell, GA: Innervation of the heart. Br Heart J, 1953,15(2);159-171.
32. Cavallotti, C, Bruzzone, P, Mancone, M: Cate-cholaminergic nerve fibers and beta-adrenergic receptors in the human heart and coronary vessels. Heart Valves 2002, 17(1);30-35.
33. Hanna, P, Rajendran, PS, Ajiola, O, Vaseghi, M, Andrew, J et al: Cardiac neuroanatomy-imaging nerves to define functional control. Auton Neurosci 2017,207;48-58.
34. Christensen, AH, Nygaard, S, Rolid, K, Ny-troen, K, Gullestad, L et al: Early signs of sino-atrial reinnervation in the transplanted heart. Transplantation 2021, 105(9);2086-2096.
35. Grupper, E, Gewirtz, H, Kushwaha, S: Reinner-vation post-transplantation. Eur J Heart, 2018, 39(20);1799-1806.
36. Crick, SJ, Wharlton, J, Sheppard, MN, Royton, D, Yacoub, MH et al: Innervation of the human cardiac conduction system: A quantitative im-munohistochemical and histochemical study. Circulation 1994, 89(4);1697-1708.
37. Sparrow, MP, Weichselbaum, M, McCry, B Jr: Development of the innervation and airway smooth muscle in human fetal lung. Am J Respir Cell Mol Biol 1999, 20(4);550-560.
38. Lauwryns, JM, Van Ranst, L: Protein gene product 9.5 expression in the lungs of humans and other mammals. Immunocytochemical de-tection in neuroepithelial bodies, neuroendo-crine cells and nerves. Neurosci 1988, 85(3);311-316.
39. Laitinen, A: Autonomic innervation of the hu-man respiratory tract as revealed by histo-chemical and ultrastructural method. Eur J Respir Dis 1985, 140(Suppl);1-42.
40. Fisher, A, Hoffmann, B: Nitric oxide synthase in neurons and nerve fibers of lower airways and in vagal sensory ganglia of man. Correlations with neuropeptides. Am J Respir Crit Med 1996, 154(1);209-216.
41. Hiayet, MA, Wiid, HA, Wilson, AS: Investiga-tions on the innervation of the human dia-phragm. Anat Rec 1974, 179(4);507-516.
42. Martin, M, Li, K, Wright, MC, Lepore, AC: Functional and morphological assessment of diaphragm innervation by phrenic motor neu-rons. J Vis Exp 2015, 99;e52605.
43. Verlinden, TJM, van Djik, P, Herrier, A, de Gierde Vries, C, Lamers WH et al: The human phrenic nerve serves as morphological conduit for autonomic nerves and innervates the caval body of the diaphragm. Scientific Rep, 2018, 8;11697.
44. Young, RL, Page, AJ, Cooper, NL, Fishby, CL, Blickshow, LA: Sensory and motor innervation of the crural diaphragm by the vagal nerve. Gastroenterol 2010, 138(3);1091-1101.
45. Gazami, BA, Srinivassan, S: Enteric nerve sys-tem in the small intestine: Pathophysiology and clinical implication. Curr Gastroenter Rep 2010, 12(5);358-365.
46. Garcia-Victoria, M, Garcia-Corchon, C, Ro-drigues, JA, Garcian-Amigot, E, Burrel, MA: Expression of neuronal nitric oxide synthase in several type of the rat gastric epithelium. J Histochem Cytochem 2000, 48(8);1111-1119.
47. Timermans, JP, Babiers, M, Scheurmann, DW, Bogers, JJ, Adriansen, D et al: Nitric oxide synthase immunoreactivity in the enteric nerv-ous system of the developing human digestive tract. Cell Tissue Res 1994, 275(2);235-245.
48. Wattchow, DA, Brooks, SJ, Cost, M: The mor-phology and projections of retrogradely la-belled myenteric neurons in the human intes-tines. Gastroenterol 1995, 109(3);866-875.
49. Porter, AJ, Wittchow, DA, Brooks, SIH, Schur-mann, M, Costa, M: Choline acetyltransferase immunoreactivity in the human small and large intestine. Gastroenterol 1996, 111(2)401-408.
50. Assouz, LL, Sharma, S: Physiology, Large intes-tine. Stat Pearls, August 9, 2021.
51. Felten, DL, Ackerman, KD, Wiegand, SJ, Fel-ten, SY: Noradrenergic sympathetic innerva-tion of the spleen. 1. Nerve fibers associate with lymphocytes and macrophages in specific compartments of the splenic white pulp. J Neu-rosci Res 1987,181(1);28-36.
52. Bujis, R, van der Villet, J, Garidodus, ML, Huntinga, I, Escobar, C: Spleen vagal dener-vation inhibits the production of antibodies to circulating antigens. PLOS 3(9);e3152,2008.
53. Hu, D, Al-Shalan, MHA, Shi, Z, Wang, P, Wu, Y et al: Distribution of nerve fibers and nerve-immune cell association in mouse spleen re-vealed by immunofluorescent staining. Sci Rep 10;9850, 2020.
54. Giogrio, R, Sternini, C, Anderson, K, Brecha, N, Go, V: Tissue distribution and innervation pat-tern of peptide immunoreactivity in the rat pancreas. Peptides 1992, 13(1);91-98.
55. Ushiki, T, Watanabe, S: Distribution and ultra-structure of the autonomic verves in the mouse pancreas. Microscop Res Tech 1997, 37(5-6);399-406.
56. Rodriguez-Diaz, R, Caicedo, A: Novel ap-proach to studying the role of innervation plays in the biology of pancreatic islets. Endo-crinol Metab Clin North Am 2013, 42(1);39-56.
57. Chandra, R, Liddle, RA: Neural and hormonal regulation of pancreatic secretion. Curr Oppin Gastroenterol 2014, 30(5);490-494.
58. Tomita, T, Lacy, PE, Matschinsky, FM, McDan-iel, M: Effects of alloxan on insulin secretion I isolated rat islets perifused in vitro. Diabetes 1974, 23(6);517-524.
59. Barthout, HR, Powley, TL: Identification of va-gal preganglion that mediates cephalic phase insulin. Am J Physiol 1990, 258(2);R523-R530.
60. Li, W, Yu, G, Liu, Y, Sha, L: Intrapancreatic ganglia and neural regulation of pancreatic endocrine secretion. Front Neurosci 13;1-10,2019.
61. Ahren, B, Taborsky, GJJr: The mechanism of vagal nerve stimulation of glucagon and insu-lin secretion. Endocrinol 1986, 118(1);1551-1557.
62. Tomita, T, Friesen, SR, Kimmel, JR, Pollock, HG: Pancreatic polypeptide-secreting islet cell tu-mors. Am J Pathol 1983, 113 (2);134-142.
63. Mudler, J, Hokfelt, T, Knuepfer, MM, Kopp, UC: Renal sensory and sympathetic nerves re- innervate kidney in a similar time-dependent fashion after renal denervation in rats. Am J Physiol Rregul Integr Physiol 2013, 304(8);R675-R682.
64. Barajas, L, Powers, K: Monoaminergic innerva-tion of rat kidney. A quantitative study. Am J Physio 259(pt2);F503-F511.
65. Mitchell, AGE: The nerve supply of the kid-neys. Acta Anat 1950, 10(1);1-37.
66. Di Bona, GF, Kopp, UC: Nerve control of renal function. Phys Rev 1997, 77(1);75-197.
67. Ahren, B: Thyroid neuroendocrinology: Nerve regulation of thyroid hormone. Endocr Rev 1986, 7(2);149-155.
68. Amenta, F, Caporuscio, D, Ferrante, F, Porcelli, F, Zomparelli, M: Cholinergic nerves in the thy-roid gland. Cell Tissue Res 1978, 195(2);367-370.
69. Grunditz, t, Hakenson, R, Rerup, C, Sundler, F, Uddman, R: Neuropeptide Y in the thyroid gland: neuronal localization and enhancement of stimulated thyroid hormone secretion. Endo-crinol 115(4);1537-1542.
70. Silva, JE, Bianco, SD: Thyroid adrenergic in-teractions: Physiological and clinical implica-tions. Thyroid 2008, 18(2);157-165.
71. Parker, TL, Kesse, WK, Mohamed, AA, Afe-work, M: The innervation of the mammalian adrenal gland. J Anat 1993, 183(pt 1);265-276.
72. Charlton, BG, Nkomazana, OF, Mc Gadey A, Neal, DE: A preliminary study of acetylcho-line-esterase-positive innervation in the human adrenal cortex. J Anat 1991, 176;99-104.
73. Holgert, H, Gagerlind, A, Hokfelt, T: Immuno-histochemical characterization of epidermic innervation of the rat adrenal. Horm Metab Res 1998, 30(6-7);3156-3222.
74. Murabayashi, H, Kuramoto, H, Kawano, H, Sasaki, M, Katamura, N et al Immunohisto-chemical feature of substance p-immunoreactive chromaffin-cells and nerve fi-bers in the rat adrenal gland. Arch Histol Cytol 2007, 70(3);183-196.
75. Heym, C, Braun, B, Shuji, Y, Klimasschewski, L, Colombo-Benkmann, M: Immunohistochemical correlation of human adrenal verve fibers and thoracic dorsal root neurons with special ref-erence to substance P. Histochem Cell Biol 1995, 104(3);233-243.
76. Fung, MM, Miveerros, OH, O’Conner, DT: Dis-eases of the adrenal medulla. Acta Physiol 2007, 192(2); 325-335.
77. Ji, RC, Kato, S: Intrinsic interrelationship of lymphatic endothelia with nerve elements in the monkey urinary bladder. Acta Rec 2000, 259(1);86-96.
78. Yoshimura, N, Chancellor, MB: Neurophysiolo-gy of the lower urinary tract function and dys-function. Rev Urol 2003, 5(Suppl 8);s3-s10.
79. de Groat, W: Integrative control of the lower urinary tract: preclinical perspective. Br J Pharmacol 2006, 147(Suppl 2);s25-s40.
80. Andersson, KE: Bladder activation: afferent mechanisms. Urol 2002, 39(Suppl 1);43-50.
81. Bauer, MM, Smith, PG: Estrogen and female reproductive tract innervation: Cellular and molecular mechanisms of autonomic neuroplas-ticity. Auton Neurosci 2015, 187(1);1-17.
82. Hoyle, CV, Stones, RW, Robson, T, Whitley, K, Burnstock, GY: Innervation of vascular and mi-crovasculature of the human vagina by NOS and neuropeptide-containing nerves. J Anat 1996, 188(3);633-644.
83. Li, T, Liau, H, Gao, X, Li, X et al: Anatomic dis-tribution of nerves and microvascular density in the human anterior vaginal wall: Prospec-tive study. PLOS One, 2014, 9(11):e110239.
84. Griebling, TL, Liao, Z, Smith, PG: Systemic and tropical hormone therapies reduce vaginal in-nervation density in postmenopausal women. Menopause 2012, 19(6);630-635.
85. Bae, SE, Corcoran, BM, Watson, DE: Immuno-histochemical study of the distribution of ad-renergic and peptidermic innervation in the equine uterus and the cervix. Reproduction 2001, 122 (2);275-282.
86. Spezio Sardo, D, Floria, A, Soss Fernandez, LM, Guerra, G, Spinelli, M et al: The potential role of endometrial nerve fibers in the patho-genesis of pain generation during endometrial biopsy at office hysteroscopy. Reprod Sci 2015, 22(1);124-131.
87. Donadio, V, Nolano, M, Porovitera, V, Stancanelli, A, Lullo, F et al: Skin sympathetic adrenergic innervation: an immunofluores-cence confocal study. Am Neurol 2006, 59(2);376-381.
88. Lauria, G, Deviglia, G: Skin biopsy as a diag-nostic tool in peripheral neuropathy. Nat Clin Prac 2007, 3(10);546-557.
89. Wang, N, Gibbons, CH, Freeman, R: Novel immunohistochemical technique using discrete signal amplification systems for human cutane-ous peripheral nerve fiber imaging. J Histo-chem Cytochem 2011, 59(4);382-390.
90. Iwami, D, Ohi, R, Nio, M, Shimaoka, S, Sano, N: Abnormal distribution of nerve fibers in the liver of biliary atresia. Tohoku J Exp Med 1997, 181(1);57-65.
91. Akiyoshi, H, Gonad, T, Terada, T: A compara-tive histochemical and immunohistochemical study of aminergic, Cholinergic and peptider-mic innervation in rat, hamster, guinea pig, dog and human liver. Liver 1998, 18(5);352-359.
92. Grant, WF, Nicol, L, Thorn, SR, Grove, KL, Friedman, JR et al: Peripheral exposure to a high-fat diet is associated with reduced hepat-ic sympathetic innervation in one-year male Japanese macaques. PLOS One 2012, 7(10):e48119.
93. Tomita, T: Localization of nerve fibers in colon-ic polyp, adenoma and adenocarcinoma by immunocytochemical staining for PGP 9.5. Dig Dis Sci 2012, 57(2);364-370.
94. Tomita, T: PGP 9.5 immunocytochemical stain-ing for pancreatic endocrine tumors. Islets 2013, 5(1);1-7.