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Home  >  Medical Research Archives  >  Issue 149  > Immunohistochemical Staining for Nerve Fibers in Normal Tissues with Frozen Sections: Structure and Function Rela-tionship
Published in the Medical Research Archives
Jul 2023 Issue

Immunohistochemical Staining for Nerve Fibers in Normal Tissues with Frozen Sections: Structure and Function Rela-tionship

Published on Jul 01, 2023




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.

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Tatsuo Tomita

With routinely formalin-fixed and paraffin- embedded tissues, specific immunohistochemical staining for nerve fiber is limited since nerve fiber antigens are expressed at low levels in the majority of normal organs except central nervous system1. Traditionally, Bielschowsky silver stain has been used to stain nerve fibers in degenerative brain dis- eases including Alzheimer’s disease2,3. Nerve fibers are mixture of myelinated and unmyelinated nerves and peripheral nerve fibers are poorly stained in the routinely H & E sections, since individual axons are commonly small individual in dimeter (1 - 5µm) and myelin sheath of myelinated nerves are color- less in the H & E sections1. Thus, small nerve fibers are often indistinguishable from fibrotic fibers in the routinely formalin-fixed and paraffin-embedded sections1. The previously reported immunohisto- chemical studies of nerve fibers in the endometrium had been performed with routinely paraffin-em- bedded tissues4-7. Epidermal peripheral nerve fi- bers had been extensively studied with skin punch biopsy using 50 µm thick floating tissue sections6-8 after being fixed in 2% paraformaldehyde and these procedures are very labor-intensive. Skin bi- opsy for the assessment of intradermal sensory nerve fiber using the pan-axonal marker, PGP 9.5 is an established clinical and research technique, and this technique may also be used to assess other population of nerve fibers in the other tissues8-11. We had previously utilized immunohistochemical staining with frozen sections for immunostaining lym- phatic and blood vessels with normal monkey tis- sues12,13 and extended this staining technique to im- munostain peripheral nerve fibers in the normal tis- sues14. The current study aimed to detect the pres- ence of nerve fibers in normal tissues including heart, lung, small intestine, large intestine, dia- phragm, spleen, pancreas, adrenal gland, kidney, urinary bladder, vagina and normally cycling en- dometrium. The most commonly used marker for nerve fibers is PGP 9.5, which is a non-specific pan- axonal marker15-17. In this study we used antibodies against neurofilament (NF) and neural cell adhesion molecule, CAM (CD56). NF antibody is a highly spe- cific immunohistochemical marker for myelinated nerve fibers, which immunostains Aβ, Adelta and B fibers, and Adelta fibers are small, myelinated fi- bers and transmit sharp, pricking localized pain to CNS18-21. C-fibers are small unmyelinated fibers and transmit dull, aching, burning poorly located pain20,21. CD56 expression is compatible to that of N-CAM and is expressed in thin nerve fibers, fine varicose and sensory nerve endings, cell membrane of ganglion cells, young, striated muscle cells, thick nerve fibers and perikaryal of ganglion cells while adult striated muscle fibers were reportedly CD56 negative22-25. Nitric oxide synthases (NOSs) are a family of enzymes catalyzing the production of ni- tric oxide from arginine26. Several cell types had been reported positive for NOS to be added as a marker for many neuroendocrine cells20-24. NOS has been added as a marker for nerve fibers and neu- roendocrine cells26,27.

Materials and Methods
Normal tissues of rhesus monkey were ob- tained at necropsy including heart, lung, small intes- tine, large intestine, diaphragm, spleen, pancreas, kidney, adrenal, thyroid, urinary bladder, vagina and cycling endometrium. The wedges of uterine tis- sue were processed from the cycling monkeys from Day 3 to Day 28. Additional monkeys were inserted with Silastic IUD (6 cm) into uterine cavity filled with an IUD containing progesterone antagonist, ZK 23011 (Leiras OY, Finland), which was kept for 5 months28. After the normal organs and uterus had been removed, a wedge of tissue from the inner en- dometrial surface to the myometrium including the full thickness of endometrium and contiguous myom- etrium was taken as described before at an aver- age tissue size of 1 x 1 x 0.4 cm12-14. Fresh wedge tissues were microwave-irradiated for 7 sec in a mi- crowave oven, embedded in OCT, frozen in liquid propane in the liquid nitrogen bath and frozen sec- tioned at 5 – 7 µm12-14. Frozen sections were mounted on Super Frost Plus slide (Fisher Scientific, Pittsburgh, PA), microwave-irradiated again on ice for 3 sec, fixed in 2% paraformaldehyde in phos- phate buffer at pH 7.4 for 10 to15 min at room temperature, and immersed twice for 2 min each in 85% ethanol12-14. Sections were incubated with blocking serum for 20 min and then with monoclonal anti-human NF (clone 2F11) and monoclonal CD56 (Dako System, Carpenteria, CA) at 1:100 dilutions overnight at 4°C, respectively. The other sections of large intestine were also immunostained with rabbit anti-PGP 9.5 (Gene Tex, TGX 17039, Irvine, CA) and rabbit anti-NOS (SAB 45020111, Millipore Sigma, Burlington, MA) at 1 : 100 dilutions to com- pare immunostaining of nerve fibers with that of NF and CD56. After rinsing and immersion in blocking serum again, sections were incubated with second antibody (1 : 200 dilutions) for 30 min at room tem- perature. Final visualization was achieved with the ABC kit (Vector Laboratories, Burlinga-me, CA) us- ing diaminobenzidine tetrahydrochloride (Dojindo Molecular Technology, Rockville, MD) for brown col- oring. Tissue sections were then lightly counter- stained with hematoxylin to facilitate identification of cellular components. Additional intestinal and kidney tissues were fixed in a mixture of 2% para- formaldehyde and 1% formalin and were embedded in paraffin. The paraffin sections were treated for antigen retrieval procedure, then the sections were stained with rabbit-anti-PGP 9.5 and rabbit anti-NOS at 1: 100m dilutions for nerve fi- bers, respectively.

Heart has rich myelinated and non-myelin- ated nerve fibers in the subepicardial connective tissue (Fig. 1-A) and a few infiltrating nerve fibers in the arterial wall of the ventricle by NF staining while CD56 immunostaining revealed more peri- arterial nerve fibers than NF immunostaining (Fig. 1-A and -B). In lung, a very few scattered non-my- elinated nerve fibers were immunostained in the peri-arterial tissue equally by both NF and CD56 immunostaining (Fig.1-C and -D). In the cross sections of diaphragm, there were abundant plexuses and myelinated nerve fibers in the fibrous stroma, from which myelinated and non-myelinated fibers infil- trated into each muscle bundles by NF immunostain- ing and less non-myelinated nerve fibers by CD56 immunostaining (Fig. 1-E and -F). Duodenum had abundant nerve plexuses and myelinated nerve fi- bers in the deep submucosa (Meissner’s plexus) and other myenteric plexuses between the inner and outer muscular layer (Auerbach’s plexus) by NF staining (Fig. 2-A). CD 56 immunostaining revealed similar findings albeit less darker staining for intra- muscular nerve bundled than NF staining (Fig. 2-B). Frozen sectioning large intestine was difficult and frozen sections were compared to the routinely par- affin-embedded sections. Frozen sections revealed multiple myelinated nerve bundles in the inner and outer smooth muscle layer with some vertically infil- trating non-myelinated nerve fibers in the inner cir- cular muscle by NF immunostaining (Fig.2-C). CD56 immunostaining also revealed numerous intramuscu- lar nerve bundles and vertically infiltrating nerve fi- bers plus non-myelinated nerves in the muscularis mucosa, some of which infiltrated into the deep mu- cosal stroma (Fig. 2-D). Immunostaining for PGP 9.5 and NOS revealed more nerve fibers than by NF in the frozen sections (Fig. 2-E and -F). By contrast, routinely paraffin-embedded sections showed less scattered submucosal nerve fibers and myelinated intermuscular nerve bundled by PGP 9.5 and NOS immunostaining (Fig. 2-G and -H). Only a few nerve fibers were stained in the circular smooth muscle layer with paraffin-embedded sections (Fig. 2-G and -H). Spleen showed a few myelinated nerve fi- bers in the germinal center of the white pulp by NF staining (Fig. 3-A) while CD56 immunostaining re- vealed not only myelinated nerve fibers in the ger- minal center but diffusely, distributed non-myelin- ated nerve fibers in the arterial wall in the white pulp (Fig. 3-B).

Pancreatic islets were faintly immunostained by NF (Fig. 3-C) and stronger im- munostained by CD56 (Fig. 3-D). Ganglion was noted close to the islets, which was surrounded by thin non-myelinated nerve fibers, some of which penetrated into the islets revealed by CD56 im- munostaining but not by NF immunostaining (Fig. 3- C and -D). The cortex of kidney showed very few myelinated nerve fibers by CD56 staining and non- myelinated nerve fibers by NF immunostaining ( Fig. 3-E and -F). The formalin-fixed and paraffin-em- bedded sections revealed large nerve bundles in the deep cortex with dense staining by PGP 9.5 but negatively stained by NOS, where scattered nerve fibers were noted plus mesangium being positively stained by PGP 9.5 (Fig. 3-G and -H). In thyroid, intrafollicular fibrous tissue contained many myelin- ated nerve fibers by NF staining, which were also immunostained by CD56 staining in addition to in- clude the numerous non-myelinated nerve fibers in- filtrating into the arterial media (Fig. 4-A and -B). In adrenal, myelinated nerve fibers were depicted in the subcapsular connective tissue and a few scat- tered non-myelinated nerve fibers were infiltrated continuo-usly through the cortex to the medulla by NF staining (Fig.4-C) while abundant non-myelin- ated nerves were located in the medulla, more by NF than CD56 staining (Fig. 4-C and -D). In urinary bladder, there were ample fragmented, mostly non-myelinated nerve fibers in suburothelial tissue and numerous, mostly myelinated nerve fibers in the smooth muscle layer accompanied with some small non-myelinated fibers by NF and CD56 im- munostaining (Fig. 4-E and -F) where smooth muscle was moderately immunostained by CD56 (Fig. 4-F). Sections of vagina revealed nerve fibers adjacent to nerve plexus by NF immunostaining and numerous non-myelinated nerves in the submucosa by CD56 immunostaining (Fig. 4-G and -H). In the cycling en- dometri-um, Day 3 endometrium revealed non-my- elinated nerve fibers by NF immunostaining and a few non-myelinated nerve fibers in the thin, sloughed off basalis compared to the much more numerous myelinated and non-myelinated nerve fi- bers in the myometrium (Fig. 5-A and -B). In Day 7 endometrium, a few non-myelinated nerve fibers were shown in the deep basalis by NF immunostain- ing (Fig. 5-C). In Day 28 endometrium, some small nerve fibers were also present in the deep and mid- dle functionalis and numerous vertically arranged nerve fibers were present in the basalis (Fig. 5-D). The endometrium from the monkeys treated with ZK (an antagonist against progesterone) for 5 months revealed thickened basalis containing abundant non-myelinated nerve fibers by NF immunostaining and more numerous nerve fibers by CD56 im- munostaining (Fig. 5-E and -F).

Heart showed myelinated and non-myelin- ated nerve fibers in sub-epicardium by NF and in- tramuscular nerve fibers by CD56 immunostaining while there were a few penetrating small nerve fi- bers in the ventricle by CD56 staining around arte- rial adventitia (Fig. 1-A and -B). The cardiac auto- nomic nervous system plays to sustain the circulation of blood28-31. Beat-to-beat heartbeat was abol- ished by parasympathetic blockade but unblocked by sympathetic blockade 28. Thus, cardiac autono- mous system regulates all the crucial functions of the heart28-31. However, little is known about the distri- bution, morphology and immunohistochemistry of the nerve fibers in the human heart (32-34). Marron et al studied human heart with confocal and fluores- cent microscopy and found nerve fibers (diameter, 6 to 10 µm) immunoreactive to myelin basic protein in the arterial endocardium and coronary sinus, which were immunoreactive to PGP 9.5, tyrosine hy- droxylase and neuropeptide Y (NPY)33. Heart transplantation resulted in complete denervation of the donor heart with loss of afferent and efferent nerve connections including a complete absence of vagal nerve33. As a result, transplanted donor heart lost vagal tone and the heartbeat averages 90 beats/  min  at  rest33.  Sympathetic  reinnervation starts 6 months after transplantation34 and evidence of reinnervation is usually found during the second year after transplantation and involves the myocar- dial muscle, sinoatrial node and coronary vessels but remains incomplete and limited many years post-transplantation34-36. In lung, a very few scat- tered non-myelinated nerve fibers were immunostained adjacent to the small arterial wall (Fig. 1-C and -D).

Figure 1. Heart, Lung and Diaphragm Several myelinated nerve fibers were depicted in the pericardium by NF im- munostaining (A) while more small, myelinated and non-myelinated nerve fibers were present in the arterial adventitia in the ventricular wall by CD56 than NF immunostaining (B). In lung, a few periarterial non-myelinated nerve fibers were depicted by NF immunostaining (C) and more peri-arterial non-myelinated nerve fibers were revealed by CD56 immunostaining (D). In diaphragm, there were solid plexus of myelinated nerve fibers and scattered non-myelinated nerve fibers by NF immunostaining (E and F) and less plexus and non-myelinated nerve fibers were revealed by CD56 immunostaining in the fibrous stroma (G and H). p: pericardium, *: peri-bronchial nerve fiber, : peri-arterial neve fiber.

Using immunohistochemical staining, human lung showed PGP 9.5 immunostained in the cyto- plasm of the subepithelial bodies and neuroendo- crine cells of the respiratory epithelium37. Nerve fi- bers containing VIP-immunoreactivity were found in the human bronchial glands38,39. Fisher and Hoffman used NOS for immunostaining nerve fibers of the human airway including trachea, large and small bronchi and bronchioles and found NOS-positive nerve fibers decreased significantly from trachea to large-diameter bronchi to small-bronchi where NOS-nerve fibers were completely absent in the bronchi40. Our finding of very few NF and CD56 immunopositive nerve fibers corresponds to their findings. The cross sections of the diaphragm re- vealed contrasting findings between NF and CD56 immunostaining: NF immunostaining showed numer- ous plexuses, from which myelinated small nerve fi- bers were ramified by CD56 immunostaining (Fig. 1-E and -F). Plexuses probably represent motor neurons and small non-myelinated nerves may rep- resent sensory nerves 41,42. The phrenic nerve con- sists of motor, sensory and sympathetic nerve fibers and provides innervation to the diaphragm and sensation to the central tendons part of the dia- phragm43. 

Vagal sensory and motor neurons inner- vate crural diaphragm and pharyngo-esophageal ligament44. Crural diaphragm-vagal afferents show distortion of the gastro-esophageal junction, while vagal motor neurons innervate both crural dia- phragm and distal esophagus 44. The frozen sections of duodenum showed intermuscular plexus, from which submucosal plexus were derived and numer- ous non-myelinated nerve fibers penetrated throu- gh the circular inner smooth muscle as immune- stained by NF and CD56 (Fig. 2-A and -B). There were many non-myelinated nerve fibers in the mus- cularis mucosa, from which a few smaller nerve fi- bers innervated into the lamina propria (Fig. 2-A and -B). We could not obtain good sections of large intestine, which also revealed the intermuscular Au- erbach’s plexuses and submucosal Meisner’s plex- uses by both NF and CD56 staining with more fine non-myelinated nerve fibers by CD56 than NF im- munostaining (Fig. 2-C and -D). Paraffin-embedded sections of large intestine showed both Auerbach’s and Meisner’s plexuses with fewer longitudinally in- filtrating small nerve fibers by PGP 9.5 immuno- staining and less nerve fibers by NOS immunostain- ing (Fig. 2-G and -H). The superior immunostaining with frozen sections was demonstrated compared to the paraffin-embedded sections regarding much more immunostained small nerve fibers in the frozen sections with CD56 presenting as the best im- munostaining, followed by PGP 9.5 and NOS and the least with NF immunostaining (Fig. 2-A to -F). PGP 9.5 is a cytoplasmic protein, which was initially isolated from brain-extract and is specific for neu- rites, neurons and cells of the diffuse neuroendo- crine system at all stages differentiation16,17. PGP 9.5 has been used as a universal marker for all sensory nerve fibers of small-diameter fibers transmit- ting pain, and large-diameter fibers transmitting proprioception and motor fibers16,17.

However, PGP 9.5 immunostaining with paraffin-embedded sec- tions did not immunostain as many small nerve fibers as compared to NF and CD56 immunostaining in the frozen sections (Fig. 2-E to -H).The submucosal plex- uses sense the lumen environment and regulate gas- trointestinal   blood   flow  and   controlling the epithelial cell function and secretion. Three classes of enteric neurons are identified including motor neurons, intrinsic primary afferent neurons and in- terneurons. Intrinsic primary afferent neurons are primary sensors and regulate the enteric neuron sys- tem that detect the chemical features of the luminal content and physical states of the small intestine45- 49. In large intestine, Auerbachs myenteric plexuses provide motor innervation to both smooth and se- cretomotor innervation to the mucosa with both par- asympathetic and sympathetic functions50. Meiss- ner’s sub-mucosal plexus has only parasympathetic fibers, which are located in the submucosa and its nerve fibers are finer than myenteric plexuses of Auerbach’s and innervates into lamina propria as seen in the photomicrographs (Fig. 2-A and -B).

The main function of the large intestine is to absorb water and any remining absorbable nutri- ents before sending the indigestible matter to the rectum. The large intestine absorbs vitamins such as thiamine, riboflavin and vitamin K that are created by colonic bacteria50. Thus, intestines are richly in- nervated for peristalsis, absorption of nutrients and water and enteric hormone homeostasis. Spleen is diffusely innervated by small, fragmented, non-my- elinated nerve fibers as revealed by CD56 im- munostaining while NF immunostaining showed only small fragmented myelinated nerve fibers in the germinal center (Fig. 3-A and -B). Sympathetic nerve fibers were immunostained by tyrosine hy- droxylase and were richly innervated in the while pulp, distributing into marginal sinus and parafollic- ular zone51. Using immunofluorescent staining for NF, nerve fibers were found in all compartments in- cluding the splenic nodules, lymphoid sheath, mar- ginal zone, trabeculae and pulp51-53. Thus, spleen is richly and diffusely innervated by small nerve fibers and the nerve fibers may contribute to the produc- tion of antibodies to the circulating antigens 52,53. Pancreas was diffusely innervated by small, non- myelinated nerve fibers in the exocrine pancreas and these small, non-myelinated nerve fibers sur- rounded the islets, some of which penetrated into the islets revealed only by CD56 immunostaining (Fig. 3-D)54-57.

Figure 2. Duodenum and Large Intestine In duodenum, there were scattered myelinated submucosal plexus (Meissner’s) and solid, myenteric nerve plexus (Auerbachs) between the inner and outer smooth muscle layers equally by NF and CD56 immunostaining (A and B). In large intestine, in addition to Meissner’s and Auerbachs plexuses, scattered mye- linated submucosal and intermuscular layer nerve fibers were depicted by NF immunostaining (C) as compared to more nerve fibers by CD56 immunostaining, some of which penetrated into the deeper lamina propria (D). There were much less nerve fibers by PGP 9.5 immunostaining (F) than by NOS immunostaining(H) in large intestine. In paraffin-embed- ded sections of large intestine, there were some submucosal plexus and abundant myenteric plexuses while there were less myelinated nerve fibers in the smooth muscular layer and there were a few non-myelinated nerve fibers in the inner circular and outer longitudinal muscular layers by both PGP 9.5 (G) and NOS (H) immunostaining by the paraffin- embedded sections. *: Meissner’s plexus, +: Auerbachs plexus

Ganglion was located in the proximity to the islets and was immunostained for both NF and CD56 with darker staining with CD56 than with NF (Fig. 3-C and -D). Insulin secretion has two phases in response to glucose infusion in vivo: the early ce- phalic phase takes place within 5 min and the sec- ond phase occurs after 20 min after glucose infusion58. In cephalic phase in vivo, the mere pres- ence of food in the mouth, which does not increase blood glucose, results in an increase of insulin secre- tion59,60. In this phase, vagal motor neurons mainly stimulate insulin secretion while sympathetic motor neurons mainly stimulate glucagon secretion59. In the perfused pancreas and perifused isolated islets in vitro, the first phase of glucose-induced insulin se- cretion is mediated through glucose receptor and glucose transporter system, through which glucose modulates insulin secretion58,61. Pancreatic polypep- tide secretion is also mediated through autonomous nerve system in the cephalic phase of secretion60.

Thus, both intrapancreatic nerve fibers and ganglia have an important influence on pancreatic hormones secretions59-62. Frozen sections of kidney cortex showed a few myelinated nerve fibers around the small arteries by CD56 immunostaining and non- myelinated fibers by NF immunostaining (Fig.3-E and -F). Kidney tissue was also fixed in a mixture of paraformaldehyde and formaldehyde and were embedded in paraffin, and these sections were im- munostained for PGP 9.5 and NOS: many myelin- ated and non-myelinated nerve fibers were noted in the medium-sized arterial wall (Fig. 3-G and -H). A few spotty positive staining was observed in the hilus of the glomerulus, corresponding to the JG ap- paratus, and small PGP 9.5 -positive nerve fibers adjacent to the small arterial wall and the collecting tubule in the deep cortex, which were not revealed by NF immunostaining (Fig. 3-G and -H).

Figure 3. Spleen, Pancreas and Kidney In spleen, there were a few myelinated nerve fibers in the germinal center by NF and CD56 immunostaining with additional abundant, small non-myelinated fibers in the arterial wall by CD56 immunostaining (A and B). In pancreas, ganglion was weakly immunostained by NF (C) and strongly immunostained by CD56 (D). CD56 immunostained diffusely scattered non-myelinated nerve fibers in the inter-acinar stroma, some of which circled and penetrated into the islets (D), which were not immunostained by NF immunostaining (C). In kidney, there were a very few myelinated nerve fibers in the peri-glomerular arterial adventitia by both NF and CD56 immu- onstaining (E and F). In the paraffin-embedded sections of kidney, large, myelinated nerve bundles were depicted adjacent to the artery by PGP 9.5 immunostaining (G), which were not immunostained by NOS (H). c: geminal center, g: ganglion, i: islet of Langerhans, n: nerve bundle.

Sympathetic nerve fibers innervate both vascular and tubular structures throughout the kidney tissue except in the inner medulla63-66. All parts of the re- nal vasculatures are innervated with the greatest density along the afferent arterioles63,65,66 as ob- served by PGP 9.5 staining in the paraffin-embed- ded sections63-65. Innervation of major renal compo- nents including blood vessels, tubules, pelvis and glomerular forms a bidirectional neural network to relay blood flow, glomerular filtration rate, tubular resorption of sodium and water and release of renin and prostaglandins, which contribute to cardiovas- cular and renal regulation66. Thus, autonomous nerve has some control on the kidney function64,65. Renal nerve activity is commonly increased in path- ophysiological conditions such as hypertension and chronic and end-stage renal diseases65,66. Interfol- licular septum of the thyroid gland contained mye- linated nerve fibers by both NF and CD56 im- munostaining (Fig. 4-A and -B). Small non-myelin- ated nerve fibers penetrated into the tunica media of artery, which was more clearly shown by CD56 immunostaining than NF immunostaining (Fig. 4-A and -B). PGP 9.5-positive nerve fibers were de- tected in the various thyroid lesions including the normal thyroid, papillary and follicular carcinoma and majority were adrenergic although minor cho- linergic innervation was detected in the interfollicu- lar stroma67. Thyroid sympathetic adrenergic nerve terminals were found at the network around the blood vessels and also as single terminals between follicles as revealed using acetylcholine esterase histochemistry68 and immunohistochemical staining for neuropeptide Y69. Sympathetic nerve activation appears to induce thyroid hormone secretion by a direct activation of norepinephrine from the in- trathyroidal sympathetic fibers70.

The cortex and medulla of adrenal gland has been traditionally re- garded as independent entities71. Adrenal gland had myelinated nerve fibers in the sub-capsule, from which a few non-myelinated nerve fibers pen- etrated into the cortex, continuously to the medulla by more staining by NF than CD56 immunostaining (Fig. 4-C and -D). There were numerous thin non- myelinated nerve frameworks in the medulla filling the entire space (Fig. 4-C and -D). The literature has shown that nerve fibers are sparse in the adrenal cortex and are confined to the vicinity of blood ves- sels72. Many nerve fibers of adrenal gland enter the gland through its hilus, and medial margin and the majority of nerve fibers enter the gland in the me- dulla, where they ramify and give off fibers that mostly terminate with chromaffin cells. Nerve fasci- cles derived from subcapsular plexuses penetrate cortex to run alongside arterioles in the cortex to the medulla73. In the subcapsular region, myelinated and non-myelinated nerve fibers were found, and terminal axons were present in zona glomerulosa, and nerve bundles were most commonly found in zona fasciculate, and axon terminals were in the close proximity to chromaffin cells 74. Using immuno- histochemical staining for substance P and NOS, Heym et al and others found substance P and NOS staining in the adrenal cortex while all other nerve fibers were noted in both cortex and medulla75,76. Adrenal medulla received sympathetic and para- sympathetic efferent and afferent innervations75,76. Adrenal medulla is modified post-ganglion neuron and preganglionic autonomic nerve fiber and se- cretes catecholamines including epinephrine, nore- pinephrine and small amount of dopamine in re- sponse to stimulation by sympathetic preganglionic neurons76. Urinary bladder showed a striking dif- ference of innervations between the suburothelial tissue and smooth muscle layer. There were mostly non-myelinated nerve fibers in suburothelial tissue by NF and CD56 immunostaining while there were mostly myelinated nerve fibers in smooth muscle layer by NF and CD56 immunostaining (Fig. 4-G and -H). Smooth muscle of urinary bladder was moderately immunostained by CD56, while smooth muscles of intestines were negatively stained by CD56 ( Fig. 4-F). CD56 is also known to stain young, striated muscle cells20. Control of bladder and ure- thral outlet is dependent on three sets of peripheral nerves: parasympathetic, sympathetic and somatic nerves that contain afferent and efferent pathway 79.

Afferent nerves innervating the bladder have Adelta and C-fiber axons while storage and void- ing reflexes are activated by mechanosensitive Adelta afferent nerves that respond to bladder dis- tention79,80. C-fibers are non-myelinated axons, which innervate mostly in the suburothelial tissue and Adelta fibers are myelinated axons, which inner- vate mostly in the smooth muscle layer78. Parasym- pathetic preganglionic nerve terminals release ac- etylcholine, which can excite various muscarine re- ceptors in bladder smooth muscle, leading to blad- der contraction77-80. Pelvic nerve afferents, which monitor the volume of bladder and the amplitude of bladder contraction, consist of small myelinated Adelta-fibers and unmyelinated C-fibers. Normal micturition reflex is mediated by myelinated Adelta-fibers in the smooth muscle layer, that re- spond to distention79. Our study immunostained mostly non-myelinated nerve fibers in the suburo- thelial tissue and myelinated nerve fibers in the mostly smooth muscle layer corresponding to the functional study of the nerve fibers in the suburothe- lial tissues and smooth muscle layer, respectively78. Sections of vagina revealed numerous non-myelin- ated and myelinated nerve fibers in the submucosa by CD56 immunostaining while nerve plexuses and many myelinated nerves were depicted in the deep connective tissue by NF immunostaining (Fig. 4-G and -H).

Figure 4. Thyroid, Adrenal Gland, Urinary Bladder and Vagina In thyroid, myelinated nerve fibers were depicted in the interfollicular stroma by NF and CD56 immunostaining (A and B) and there were penetrating, abundant non- myelinated nerve fibers in the arterial wall by CD56 immunostaining (B). In adrenal gland capsule, there were myelin- ated nerve fiber, and in the cortex, there were a few non-myelinated nerve fibers and numerous non-myelinated nerve fibers in the medulla by NF immunostaining (C). CD56 immunostaining showed a few myelinated nerve fibers in the capsule and in the deep cortex, and many non-myelinated nerve fibers in the medulla (D). In urinary bladder, scattered mostly non-myelinated nerve fibers in the suburothelial tissue and abundant, mostly myelinated nerve fibers in the smooth muscle layer by both NF and CD56 immunostaining (E and F). Smooth muscle in the urinary bladder was mod- erately stained only by CD56 (F). Vagina showed numerous myelinated nerve plexus in the deep connective tissue by NF immunostaining (G) while several non-myelinated nerve bundles and scattered non-myelinated periarterial non- myelinated nerve fibers were present in the superficial submucosa by CD56 immunostaining (H). c: capsule, m: medulla, *: interfollicular nerve fibers, +: periarterial nerve fiber.

Functional studies provided evidence that sympa- thetic nerves were excitatory to vagal non-vascular smooth muscle while NOS-positive nerves appeared to mediate muscle relaxation and vasodilation81. With formalin-fixed and paraffin-embedded sections of human vagina, Hoyle et al and others found the relative density of nerve markers as fol- lows: PGP 9.5 > vasoactive intestinal polypeptide (VIP) > NOS81. Li et al studied nerve fibers from the biopsied different locations of human vagina using immunohistochemical staining for PGP 9.5 with for- malin-fixed and paraffin-embedded tissues82. They found a considerable difference in nerve distribu- tion in the human vagina: vaginal innervation was observed in the lamina propria and muscle layer of the anterior vaginal wall. The distal wall of the an- terior vaginal wall had significantly richer small verve fibers in the lamina propria than the proximal third and in the virginal muscle layer 83. More re- cently, Griebling et al fixed vaginal biopsy speci- men in Zamboni solution at 4°C and washed for 7 days with PBS at pH 7.4 and stored at -80°C until frozen sectioned at 10 µm. Women not receiving hormone therapy showed relatively high levels of innervation by tyrosine hydroxylase and VIP im- munostaining than women receiving hormone ther- apy84. The presence of nerve fibers in the endome- trium has been debated. In most mammalian spe- cies, the endometrium is poorly innervated by auto- nomic and efferent nerve fibers, and these nerves are associated with blood vessels85. Sympathetic nerves approach endometrial glands, suggesting a role in endometrial secretion.

Parasympathetic nerves rarely penetrate deeply in the endometrium and do not associate with endometrial glands83. Our results of nerve fibers in the secretory phase endometrium may support growing nerve fibers in the functionalis of secretory phase from basalis, which may have a role in endometrial secretion and pain during menstrual cycle. In rhesus monkey endo- metrium, fragmented non-myelinated nerve fibers were present in the basalis of in Day 3 cycling en- dometrium by NF immunostaining (Fig. 5-A). In Day 7 proliferative phase, there were some longitudinal, small nerve fibers in the basalis but not in the thin functionalis, and there were a few small nerve fi- bers at the deep functionalis, suggesting that nerve fibers grew from the basalis into functionalis in Day 7 to Day 21 (Fig. 5-B and -C). In Day 28 endome- trium during the menstrual cycle, new nerve fibers penetrated into mid-portion of enlarging function- alis (Fig. 5-C and -D). The ZK treated monkeys for 5 months, which showed extremely thickened ba- salis, revealed more, small nerve fibers by CD56 immunostaining than by NF immunostaining (5-E and -F). In the cycling human endometrium, from prolif- erative to secretory phase, the growing nerve fibers were observed from basalis to lower functionalis and then, into deeper functionalis to upper function- alis using alkaline phosphatase as the final visuali- zation for NF and CD5613. Tokushige et al used for- malin-fixed and paraffin-embedded human endo- metrial sections using PGP 9.5, NF, VIP, substance P, tyrosine hydroxylase and others, and studied the distribution of nerve fibers in the human endome- trium from women with endometriosis4.

They found more nerve fibers in the functionalis from women with endometriosis, which decreased by hormonal treatment4,5,7. There was no mention on menstrual cycle of tissue studied for endometriosis. If our re- sults were right, nerve fibers grow in the secretory phase of menstrual cycle. A similar study was also reported by Boker et al with routinely paraffin-em- bedded sections using PGP 9.5, VIP and substance P5. These authors published limited, small tissue ar- eas of positively stained photomicrographs without presenting low power photomicrographs, possibly selecting positively stained microscopic areas. With routinely paraffin-embedded tissues, reliable im- munohistochemical staining for nerve fibers is tech- nically difficult since nerve fiber antigens are ex- pressed at low levels in the majority of normal or- gans except brain and spinal cord2,3. With human hysterectomy specimen, which had been fixed in Bouin’s fixative and paraffin-embedded sections, Sparzio Sardo et al performed immunohistochemi- cal staining using S-100, neuron specific enolase (NSE) and others as nerve markers: nerve fibers were depicted at the level of functionalis and ad- nomyosis86. Adequate immunostaining for nerve fi- bers was only feasible with frozen tissues, which were frozen sectioned and were stained for nerve fiber markers as described in the nerve fibers in the skin as described below. Skin punch biopsy with the assessment of intradermal sensory nerve fibers us- ing pan-axonal marker, PGP 9.5 is an established clinical and research technique, and this technique may also be used to assess other population of nerve fibers in other tissues87,88. Another method uses initially fixed tissues, which are later frozen sectioned and the floating 50 µm sections were im- munostained for nerve fibers markers to increase the visibility of the scarcely innervated nerve fibers since thicker tissue sections increase the chance to observe denser nerve fibers (89). The endometrium, especially those of the secretory phase, is so edem- atous and fragile that the fixed floating 50 µm sec- tions are extremely difficult to process for immuno- histochemical staining for frozen sectioning at our hand. The advantages of using frozen sections for immunohistochemical staining were depicted espe- cially in the sections of intestines, diaphragm and pancreas. In intestines, fine motor nerve fibers were clearly and abundantly demonstrated at such a level compared to less nerve fibers immunostained in the paraffin-embedded sections (Fig. 2-A to -H).

Figure 5. Endometrium Day 3 endometrium showed thin sloughed off basalis where there were non-myelinated nerve fibers in the deep basalis and myometrium (A). In Day 7 endometrium, there were few non-myelinated nerve fibers at the deep basalis and a very few nerve fibers in the middle-basalis (B). In Day 21 enlarging endometrium, there were more increased nerve fibers in the deep basalis plus a few nerve fibers in the mid-basalis (C), which was immunostained using alkaline phosphatase method. In Day 28 more enlarging endometrium, there were more nerve fibers in mid basalis and a few in the enlarged deep functionalis (D). Day 3 to Day 28 endometrium was immunostained by NF. Endometrium from monkeys treated with ZK for 5 months revealed much thickened basalis containing numerous non- myelinated nerve fibers in the entire basalis as much as in the myometrium by NF immunostaining (E) while there were much more non-myelinated nerve fibers revealed in the entire thickness of the basalis by CD56 immunostaining than NF immunostaining (F). *: nerve fibers in mucosa, : nerve fibers in myometrium.

In pancreas, ample, delicate fine nerve fibers were extensively depicted in the frozen sections, some of which surrounded the islets and penetrated into the islets in a proximity of ganglion by CD56 but not by NF immunostaining (Fig. 3-C and -D), which had not been depicted in the paraffin-embedded sec- tions. NF has been widely used for immunohisto- chemical staining for nerve cells and nerve fibers, but CD56 has not been extensively used for nerve fibers. CD56 is an excellent marker for nerve fibers, sensory nerve binding and ganglion cells20. Iwami et al immunostained nerve fibers around the branches of hepatic arteries, portal veins and bile ducts in the portal area using CD5690.

Using tyrosine hydroxylase, NPY and VIP as nerve fiber markers, Ariyoshi et al also detected nerve fibers in the proximity of hepatic arteries, portal veins and bile ducts with formalin-fixed and paraffin-embed- ded sections of human liver91. More recently, Grant et al observed nerve fibers in the macaque liver in close contact with portal triads, central veins and parallel with liver sinuses92. Thus, nerve fibers are diffusely distributed in the liver as similarly diffusely observed in the spleen and pancreas (Fig. 3-A to - D). The hindside of frozen section immunostaining is that this procedure is labor-intensive and cumber- some and further, only small tissues (1 x 1 x 0.4 cm) are adequately processed in our hand12-14.

During the freezing process, the tissue may break or crack, the larger tissues tend to crack more often than the smaller tissues. The frozen tissue sections mounted on the glass slide may degenerate when they are stored for some time even stored at -70°C, so, the frozen sections should be used for immunostaining as soon as possible. For a practical purpose in de- tecting nerve fibers at a diagnostic pathology la- boratory, PGP 9.5 immunostaining with formalin- fixed and paraffin-embedded sections would be adequate for routine diagnosis for general ana- tomic pathologists 93,94.

Frozen section immunohistochemical staining has validated this technique’s superiority over the formalin-fixed and paraffin-embedded tissue sec- tions for immunostaining nerve fibers as also this techniques superiority has been previously proved for immunostaining lymphatic and blood vessels (11-13). The new findings using frozen sections im- munostaining will eventually shed light more on the basic histology and histopathology in normal and pathological tissues.

Conflicts of Interest Statement: There are no con- flicts of interest in this project since I have no com- mercial interest on this project.

Funding Statement: I had been retired from aca- demic institutions since 2003 and used my time and funded my own research as a volunteer scientist at the Oregon Health and Science University, Portland, OR, USA.

Acknowledgement: I want to express my sincere thanks to Drs Robert M Brenner and Ov D Slayden for kindly allowing me to use normal tissues of rhe- sus monkey at their research laboratory of the Or- egon National Primate Research Center, Beaverton, OR, USA. Their critical advice and comments are highly appreciated, which prompted me to pursue this project.

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