- Email: [email protected]
Histopathologic examination of resected nerves from children with anterior cutaneous nerve entrapment syndrome: clues for pathogenesis?
Journal Pre-proof Histopathologic examination of resected nerves from children with anterior cutaneous nerve entrapment syndrome: clues for pathogenesis?
Jasper Markus, Maurits van Montfoort, Justin R. de Jong, Sjoerd A. de Beer, Eleonora M.A. Aronica, Ramon R. Gorter PII:
S0022-3468(20)30152-4
DOI:
https://doi.org/10.1016/j.jpedsurg.2020.01.060
Reference:
YJPSU 59629
To appear in:
Journal of Pediatric Surgery
Received date:
7 December 2019
Revised date:
27 January 2020
Accepted date:
28 January 2020
Please cite this article as: J. Markus, M. van Montfoort, J.R. de Jong, et al., Histopathologic examination of resected nerves from children with anterior cutaneous nerve entrapment syndrome: clues for pathogenesis?, Journal of Pediatric Surgery(2020), https://doi.org/10.1016/j.jpedsurg.2020.01.060
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier.
Journal Pre-proof
Histopathologic examination of resected nerves from children with anterior cutaneous nerve entrapment syndrome: Clues for pathogenesis? Jasper Markus BSca, Maurits van Montfoort MD PhD b, Justin R. de Jong MD PhDa, Sjoerd A. de Beer MDa, Eleonora M.A. Aronica MD PhDb, Ramon R. Gorter MD PhDa a
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Emma Children's Hospital, Amsterdam UMC, University of Amsterdam & Vrije Universiteit Amsterdam, Department of Pediatric Surgery, Amsterdam, The Netherlands b Department of Neuropathology, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands
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Jasper Markus, BSc Department of Pediatric Surgery Emma Children’s Hospital Amsterdam UMC, University of Amsterdam P.O. Box 22660 1100 DD, Amsterdam, The Netherlands E-Mail: [email protected] Telephone: +31 20 444 2424
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Corresponding Author:
Jo
ur
Declarations of Interest: None
Journal Pre-proof
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Abstract Background: Anterior cutaneous nerve entrapment syndrome (ACNES) has been described as a possible cause for chronic pain in the pediatric population. However, the exact pathophysiology of ACNES is unknown. It may be caused by compression or traction of cutaneous nerve branches of intercostal nerves, or it may be the result of an infection. Therefore, we present histopathological evidence to determine the pathophysiology of ACNES. Methods: A total of seven pediatric patients underwent a neurectomy for ACNES. All specimens were sent for histopathological evaluation, including immunohistochemical staining, to evaluate if there were any signs of infection, inflammation or compression. Results: Seven out of seven (100%) histopathological specimens showed non-specific nerve degeneration. Immunohistochemical evaluation showed there were several CD68-positive macrophages present in the specimens. Four out of seven (57%) specimens showed the presence of a few CD3-positive T-cells, however, this was not suggestive for inflammation or infection. Conclusion: Our study supports the hypothesis that ACNES is caused by compression of the nerves rather than inflammation.
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Keywords: ACNES; abdominal pain; surgery; pathology Level of evidence: III
Journal Pre-proof
Introduction Recently, anterior cutaneous nerve entrapment syndrome (ACNES) has been described as a possible cause for chronic abdominal pain in the pediatric and adult population. While the exact incidence of ACNES in the pediatric population remains unknown, ACNES was identified in 13% of children originally diagnosed with functional abdominal pain.[1] Due to the increased awareness, more and more children are being referred to the (pediatric) surgeon
ro of
to evaluate whether or not ACNES might be the cause of the chronic abdominal pain.
The diagnosis of ACNES is based upon patient history and physical examination. Pain is the
-p
most dominant symptom. In addition, coughing, body stretching, or any type of movement or
re
more intense use of abdominal muscles may aggravate the pain.[2] At physical examination,
lP
painful abdominal palpation, positive pinch test and Carnett’s sign may be found.[2,3] The Carnett’s sign helps to distinguish if the pain originates from the abdominal wall or from
na
abdominal viscera. During testing for the Carnett’s sign, the investigator palpates the point of
ur
maximum pain, after which the patient is asked to tense abdominal muscles followed again by palpation at the point of maximum pain. If the pain increases during palpation with flexed
Jo
abdominal muscles, the Carnett’s sign is positive indicating the pain originates from the abdominal wall.[4] Furthermore, altered skin sensibility demonstrated by a swab or alcoholsoaked gauze strongly support the diagnosis.[2,3,5] In addition, one can inject a local anesthetic into the trigger point with or without corticosteroids and with or without ultrasound guidance.[2,6–8] Pain relief for several hours or days confirms the diagnosis.[2,5,9] If multiple injections are only successful for a short period of time, a neurectomy should be considered.[2,10,11]
Journal Pre-proof However, the exact pathophysiology of ACNES is unknown. It is postulated that the abdominal pain is caused by compression or traction of cutaneous nerve branches of intercostal nerves (Th7-Th12).[2,6,12] Although other authors hypothesized that the neuralgia might be caused secondary to respiratory infections or subclinical infection with herpes zoster.[2,4,13] Therefore, the goal of this present study was to determine if nerves resected from children with ACNES show any signs of inflammation or infection, and
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thereby supporting the role of inflammation in the pathogenesis of ACNES.
1. Methods
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1.1 Setting
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In compliance with local ethical board guidelines for clinical studies, we performed a pilot prospective cohort study at the department of pediatric surgery of Emma Children’s Hospital.
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Emma Children’s Hospital is a tertiary referral center, is an expert center for children with
na
chronic abdominal pain, and participates in the national network of surgeons treating patients with ACNES. Our team consist of three pediatric surgeons. The surgery was performed by at
Jo
ur
least two surgeons of this team.
1.2 Population and design
Children younger than 18 years who were diagnosed and treated between December 2017 and June 2018 for ACNES by one of the three pediatric surgeons from our ACNES team were eligible for inclusoin. Diagnosis of ACNES was based upon the following predefined criteria: 1. Medical history 2. Physical examination: positive Carnett’s sign 3. Positive reaction on a diagnostic injection with a local anesthetic agent.
Journal Pre-proof If a patient fulfilled the diagnostic criteria, they were treated for ACNES according to our local protocol which consists of a free-hand injection with a local anesthetic (i.e. lidocaine or bupivacaine), but without corticosteroids into the trigger point, up to a maximum of 3 times. If pain persisted after these injections, an anterior neurectomy was performed. Only those patients that underwent an anterior neurectomy in the aforementioned time period were included in this study. Patients were excluded if no nerve tissue was present during
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histopathological examination.
1.3 Surgical procedure
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The procedures were performed in a day care surgery setting by a team of two of three
re
surgeons of the ACNES team. Preoperatively, the area of maximal pain was identified and marked (together with the patient) prior to the general anesthesia. The anterior sheath of the
lP
abdominal rectus muscle was exposed via a transverse 5 cm skin incision. Dissection of the
na
subcutaneous fat tissue and Scarpa’s fascia was done with a combination of blunt and cautery dissection. All neurovascular bundles penetrating the fascial foramina of the anterior sheath
ur
were identified within a 5 cm radius. The fascial foramina were widened and the
Jo
neurovascular bundles were resected beyond the fascia after injection of a local anesthetic agent. Subsequently, the resected were sent for routine histopathological examination. All specimens sent for histopathological examination in this time period underwent standard protocol analysis including immunohistochemical staining. Nerves were pinned and marked (skin side, fascia penetration and underneath fascia site). Fresh tissue was directly sent to the department of pathology for fixation.
1.4 Fixation and slides
Journal Pre-proof The tissue samples were fixed overnight in a 4 % formaldehyde and routinely processed into liquid paraffin. Sections were cut in 4-6 µm with a microtome (Microm, Heidelberg, Germany), and mounted on positively charged slides (Superfrost + Menzel, Germany). Each specimen was histopathologically examined using haematoxylin & eosin (H & E) and luxol fast blue (LFB). An immunohistochemical examination of all surgical specimens was performed using the following panel of antibodies: S100 (1:4000, clone Z0311, DAKO, Glostrup, Denmark, pan neural marker), anti-CD68 (mouse monoclonal, clone PG-M1;
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DAKO; 1:200, monocytes, macrophages, microglia) and anti-CD3 (mouse monoclonal, clone F7.2.38; DAKO; 1:200, T lymphocytes). The slides were air dried overnight at 37°C. All
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immunohistochemical stainings were performed with a Ventana semiautomated staining
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machine (Benchmark ULTRA; Ventana, Illkirch, France) and the Ventana DAB staining
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system according to the manufacturer’s protocol.
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1.5 Outcome measures 1.5.1 Primary outcome
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Our primary outcome is defined as the number of patients with signs suggestive for
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inflammation or infection defined as: -
Positive H & E staining: influx of lymphocytes or neutrophils
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Immunohistochemistry: influx of CD3-positive T-lymphocytes
The histopathological assessment was performed by two neuropathologists, who determined if there were any signs of inflammation or infection.
1.5.2. Secondary outcome Our secondary outcomes were treatment success, defined as less pain or no pain after neurectomy, and complication rate during and after surgery.
Journal Pre-proof
1.6 Statistical methods Due to the nature of this study, only descriptive analysis was performed. Primary and secondary outcomes were reported as n (%).
2. Results 2.1 General characteristics
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In this time period, a total of eight children underwent anterior neurectomy, although one patients was excluded from this study due to the fact that on histopathological examination
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no nerve tissue could be identified. General characteristics of the seven included patients are
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shown in table 1, with a median (range) age of 15 (14-16) years old. Six patients (86%) were female, and one patient (14%) had a medical history of abdominal surgery. None of the
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duration was 47 (19-280) days.
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patients showed any signs of skin disease or infection. The median (range) follow-up
2.2 Primary outcome
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2.2.1 Histopathological examination
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All histopathological specimens showed non-specific nerve degeneration. H&E staining did not show any signs of infection or inflammation (see table 2) (see figure 1).
2.2.2 Immunohistochemistry All histopathological specimens were S100 positive, confirming the presence of nerve tissue. There were several CD68-positive macrophages present in the specimens. Four out of seven (57%) specimens showed the presence of a few CD3-positive T-cells, however, this was not suggestive for inflammation (see table 2) (see figure 1).
Journal Pre-proof 2.3 Secondary outcome All seven patients (100%) were pain free after the neurectomy during the follow-up period.
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There were no peri- and postoperative complications.
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3. Discussion This study shows that there is no influx of CD3-positive cells (lymphocytes) and only limited influx of CD68-positive cells (macrophages) in the resected nerves of children undergoing a neurectomy for ACNES. These findings do not support the hypothesis that inflammation or infection might play a role in the pathogenesis of ACNES as postulated in the past by some authors. They stated that the neuralgia in patients with ACNES might be the result of
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secondary inflammation of the nerves due to viral infections.[2,4,13] Results, however, should be interpreted with care as this was a pilot study with only a limited number of
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patients.
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Varicella zoster virus (VZV), an alpha herpesvirus only found in humans, causes varicella
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(chickenpox) upon primary infection.[14–16] After the infection resolves, VZV becomes latent in peripheral nervous system (PNS) ganglia of nearly all previously infected
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individuals with no corresponding morphological changes.[16] After reactivation from a
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latent state it causes herpes zoster (HZ).[14–16] It has been shown that some neurons do not survive after an episode of HZ and that axonal injury, demyelination and inflammation occur
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from the dorsal root to the skin.[17,18] After HZ resolves, many patients experience severe persistent pain (postherpetic neuralgia; PHN), which can persist for months up to years.[14– 16,19] Postmortem microscopic analysis of ganglia in patients with known HZ infection showed the presence of inflammatory cells around neurons.[16,20,21] Since none of the patients showed any signs of skin infection, no signs of infection or inflammation was found during microscopic evaluation and no large presence of CD68-positive histiocytes or influx of CD3-positive T-cells, it is unlikely that inflammation or infection of the nerves plays an important role in ACNES.
Journal Pre-proof Our findings support the hypothesis that ACNES is caused by compression or traction of cutaneous nerve branches of intercostal nerves (Th7-Th12), or by herniation of connective fat tissue surrounding the nerve within the neurovascular channel in the rectus muscle leading to nerve ischemia and pain.[2,6,12] This is reflected by the limited influx of CD68-positive cells with concomitant nonspecific degenerative signs that are also present in other known nerve compression syndromes, of which carpal tunnel syndrome (CTS) is the most common. CTS is caused by traction and compression of the median nerve, causing disorders of the
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intraneural microcirculation, resulting in nerve edema, demyelination and degeneration from the site of compression and beyond.[22] Another nerve compression syndrome is meralgia
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paresthetica. Meralgia paresthetica is caused by compression of the lateral femoral cutaneous
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nerve[23], and causes similar symptoms as ACNES, including pain and numbness.[24] Histopathological evaluation of resected nerves shows reduced fiber density with multifocal
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fiber loss, loss of myelinated fibers, perineurial thickening, Renaut bodies and subperineural
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edema.[25] These findings have also been shown in other nerve compression syndromes, including human ulnar nerves at sites of compression.[26] Accordingly, we found non-
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specific degeneration of nerve fibers, indicated by the CD68-positive histiocytes within the
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nerve bundles, supporting the fact that the degenerative changes are likely to be caused by compression or traction.
To our knowledge, this is the first study reporting the histopathological findings of nerves resected from children diagnosed with ACNES. Still, several limitations are present. One potential limitation is the limited number of patients included in this study. Although it is possible that this might reduce the viability of our conclusion, we still could not find any suggestion of inflammation in eight consecutive patients. Secondly, it remains unclear whether or not the injection with a local anesthetic agent could influence our results. In our
Journal Pre-proof opinion, injection with a local anesthetic could not be of any influence on the degree of inflammation, although it might cause non-specific degenerative changes. Since we did not use corticosteroids in our injection, it is highly unlikely that the injection had any effect on inflammation. Thirdly, there is the possibility of a sampling error. However, the painful area was marked together with the patient before the operation and all nerves encountered in this area were sent for histopathological examination. We therefore believe that the resected
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nerves were representative for the pediatric population.
4. Conclusion
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Our study supports the hypothesis that ACNES is caused by compression of the nerves rather
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than inflammation or infection.
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Acknowledgements
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There are no acknowledgements. This research did not receive any specific grant from
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funding agencies in the public, commercial, or not-for-profit sectors.
Journal Pre-proof Figure Legend Figure 1. Characteristic findings in nerve biopsies from patients with ACNES who underwent neurectomy. (a) H&E staining shows nerve fiber (pink) surrounded by adipose tissue (white) and no influx of either neutrophils or lymphocytes as sign of infection or inflammation. (b) S100 staining confirms the presence of neural tissue. (c) CD68 staining shows the presence of several macrophages (brown cells). (d) CD3 staining
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shows the presence of a single T-lymphocyte (indicated by the arrow).
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References [1] Siawash M, De Jager-Kievit JWA, Ten WTA, Roumen RM, Scheltinga MR. Prevalence of anterior cutaneous nerve entrapment syndrome in a pediatric population with chronic abdominal pain. J Pediatr Gastroenterol Nutr 2016;62:399–402. [2] Scheltinga MR, Roumen RM. Anterior cutaneous nerve entrapment syndrome (ACNES). Hernia 2018;22:507–16. [3] Siawash M, Roumen R, Ten WTA, van Heurn E, Scheltinga M. Diagnostic characteristics of anterior cutaneous nerve entrapment syndrome in childhood. Eur J Pediatr 2018;177:835–9. [4] Carnett JB. Intercostal neuralgia as a cause of abdominal pain and tenderness. Surg Gynecol Obstet 1926;42:625–32. [5] Boelens OB, Scheltinga MR, Houterman S, Roumen RM. Management of anterior cutaneous nerve entrapment syndrome in a cohort of 139 patients. Ann Surg 2011;254:1054–8. [6] Kifer T, Mišak Z, Jadrešin O, Hojsak I. Anterior cutaneous nerve entrapment syndrome in children: A prospective observational study. Clin J Pain 2018;34:670–3. [7] Siawash M, Mol F, Tjon-A-Ten W, Perquin C, van Eerten P, van Heurn E, et al. Anterior rectus sheath blocks in children with abdominal wall pain due to anterior cutaneous nerve entrapment syndrome: a prospective case series of 85 children. Paediatr Anaesth 2017;27:545–50. [8] Sahoo RK, Nair AS. Ultrasound Guided Transversus Abdominis Plane Block for Anterior Cutaneous Nerve Entrapment Syndrome. Korean J Pain 2015;28:284–6. [9] Boelens OBA, Scheltinga MR, Houterman S, Roumen RM. Randomized clinical trial of trigger point infiltration with lidocaine to diagnose anterior cutaneous nerve entrapment syndrome. Br J Surg 2013;100:217–21. [10] Siawash M, Maatman R, Tjon A. Ten W, van Heurn E, Roumen R, Scheltinga M. Anterior neurectomy in children with a recalcitrant anterior cutaneous nerve entrapment syndrome is safe and successful. J Pediatr Surg 2017;52:478–80. [11] Armstrong LB, Dinakar P, Mooney DP. Neurectomy for anterior cutaneous nerve entrapment syndrome in children. J Pediatr Surg 2018;53:1547–9. [12] Akhnikh S, De Korte N, De Winter P. Anterior cutaneous nerve entrapment syndrome (ACNES): The forgotten diagnosis. Eur J Pediatr 2014;173:445–9. [13] Davis JH. Segmental neuralgia in childhood simulating visceral disease. JAMA 1936;107:1620–6. [14] Kinchington PR, Goins WF. Varicella zoster virus-induced pain and post-herpetic neuralgia in the human host and in rodent animal models. J Neurovirol 2011;17:590–9. [15] Gilden D, Nagel M, Cohrs R, Mahalingam R, Baird N. Varicella Zoster Virus in the Nervous System. F1000Research 2015;4. doi:10.12688/f1000research.7153.1. [16] Kleinschmidt-DeMasters BK, Gilden DH. Varicella-zoster virus infections of the nervous system: Clinical and pathologic correlates. Arch Pathol Lab Med 2001;125:770–80. [17] Petersen KL, Rice FL, Farhadi M, Reda H, Rowbotham MC. Natural history of cutaneous innervation following herpes zoster. Pain 2010;150:75–82. [18] Watson C, Deck JH. The neuropathology of herpes zoster with particular reference to postherpetic neuralgia and its pathogenesis. In: Watson C, editor. Herpes zoster and postherpetic neuralgia, Amsterdam: Elsevier Inc.; 1993, p. 139–58. [19] Gilden DH, Kleinschmidt-DeMasters BK, LaGuardia JJ, Mahalingam R, Cohrs RJ. Neurologic Complications of the Reactivation of Varicella–Zoster Virus. N Engl J Med 2000;342:635–45. [20] Watson CPN, Deck JH, Morshead C, Van der Kooy D, Evans RJ. Post-herpetic
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[26]
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[25]
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[24]
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[23]
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[22]
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[21]
neuralgia: Further post-mortem studies of cases with and without pain. Pain 1991;44:105–17. Smith FP. Pathological studies of spinal nerve ganglia in relation to intractable intercostal pain. Surg Neurol 1978;10:50–3. Aboonq MS. Pathophysiology of carpal tunnel syndrome. Neurosciences (Riyadh) 2015;20:4–9. de Ruiter GCW, Lim J, Thomassen BJW, van Duinen SG. Histopathologic changes inside the lateral femoral cutaneous nerve obtained from patients with persistent symptoms of meralgia paresthetica. Acta Neurochir (Wien) 2019;161:263–9. Cheatham SW, Kolber MJ, Salamh PA. Meralgia paresthetica: a review of the literature. Int J Sports Phys Ther 2013;8:883–93. Berini SE, Spinner RJ, Jentoft ME, Engelstad JK, Staff NP, Suanprasert N, et al. Chronic meralgia paresthetica and neurectomy: a clinical pathologic study. Neurology 2014;82:1551–5. Neary D, Eames RA. The pathology of ulnar compression in man. Neuropathol Appl Neurobiol 1975;1:69.
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Table 1. Patient characteristics General characteristics Age (years) 15 (14-16) Female. No (%) 6 (86) Previous abdominal surgery. 1 (14) No (%) Skin disease or infection 0 (0) during physical examination. No (%) Data displayed as median (range), unless stated otherwise
Journal Pre-proof Table 2. Histopathological changes in nerves of children with ACNES Nerve tissue
CD68positive
CD3positive
Microscopic histopathological evaluation Fibro-adipose tissue with nerve fibers showing degenerative changes. No inflammation. Nerve tissue with degenerative changes. No inflammation.
1
Yes
Yes
No
2
Yes
Yes
Yes
3
Yes
Yes
Yes
Fibro-adipose tissue with nerve fibers. No inflammation. Vascular structures show hemorrhagic changes.
4
Yes
Yes
No
5
Yes
Yes
No
6
Yes
Yes
Yes
Fibro-adipose tissue with nerve fibers showing degenerative changes. No inflammation. Fibro-adipose tissue with nerve fibers showing degenerative changes. No inflammation. Fibro-adipose tissue with nerve fibers showing degenerative changes. No inflammation.
7
Yes
Yes
Yes
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Fibro-adipose tissue with nerve fibers showing degenerative changes. No inflammation.
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Immunohistochemistry
Conclusion
CD68 shows the presence a few macrophages.
No signs of inflammation
CD68 shows the presence of macrophages in the nerve fibers. A few CD3-positive T-cells are present, without aggregation. CD68 shows the presence of macrophages in the nerve fibers. A few CD3-positive T-cells are present, however, not around the nerve fibers. CD68 shows the presence of a few macrophages in the nerve fibers. CD68 shows the presence of a few macrophages around the nerve fibers. CD68 shows the presence of a few macrophages around the nerve fibers. A few CD3-positive T-cells are present. CD68 shows the presence of a few macrophages around the nerve fibers. A few CD3-positive T-cells are present.
No signs of inflammation
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Patient
No signs of inflammation
No signs of inflammation
No signs of inflammation
No signs of inflammation
No signs of inflammation
Figure 1
Jasper Markus, Maurits van Montfoort, Justin R. de Jong, Sjoerd A. de Beer, Eleonora M.A. Aronica, Ramon R. Gorter PII:
S0022-3468(20)30152-4
DOI:
https://doi.org/10.1016/j.jpedsurg.2020.01.060
Reference:
YJPSU 59629
To appear in:
Journal of Pediatric Surgery
Received date:
7 December 2019
Revised date:
27 January 2020
Accepted date:
28 January 2020
Please cite this article as: J. Markus, M. van Montfoort, J.R. de Jong, et al., Histopathologic examination of resected nerves from children with anterior cutaneous nerve entrapment syndrome: clues for pathogenesis?, Journal of Pediatric Surgery(2020), https://doi.org/10.1016/j.jpedsurg.2020.01.060
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier.
Journal Pre-proof
Histopathologic examination of resected nerves from children with anterior cutaneous nerve entrapment syndrome: Clues for pathogenesis? Jasper Markus BSca, Maurits van Montfoort MD PhD b, Justin R. de Jong MD PhDa, Sjoerd A. de Beer MDa, Eleonora M.A. Aronica MD PhDb, Ramon R. Gorter MD PhDa a
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Emma Children's Hospital, Amsterdam UMC, University of Amsterdam & Vrije Universiteit Amsterdam, Department of Pediatric Surgery, Amsterdam, The Netherlands b Department of Neuropathology, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands
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Jasper Markus, BSc Department of Pediatric Surgery Emma Children’s Hospital Amsterdam UMC, University of Amsterdam P.O. Box 22660 1100 DD, Amsterdam, The Netherlands E-Mail: [email protected] Telephone: +31 20 444 2424
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Corresponding Author:
Jo
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Declarations of Interest: None
Journal Pre-proof
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Abstract Background: Anterior cutaneous nerve entrapment syndrome (ACNES) has been described as a possible cause for chronic pain in the pediatric population. However, the exact pathophysiology of ACNES is unknown. It may be caused by compression or traction of cutaneous nerve branches of intercostal nerves, or it may be the result of an infection. Therefore, we present histopathological evidence to determine the pathophysiology of ACNES. Methods: A total of seven pediatric patients underwent a neurectomy for ACNES. All specimens were sent for histopathological evaluation, including immunohistochemical staining, to evaluate if there were any signs of infection, inflammation or compression. Results: Seven out of seven (100%) histopathological specimens showed non-specific nerve degeneration. Immunohistochemical evaluation showed there were several CD68-positive macrophages present in the specimens. Four out of seven (57%) specimens showed the presence of a few CD3-positive T-cells, however, this was not suggestive for inflammation or infection. Conclusion: Our study supports the hypothesis that ACNES is caused by compression of the nerves rather than inflammation.
Jo
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na
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re
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Keywords: ACNES; abdominal pain; surgery; pathology Level of evidence: III
Journal Pre-proof
Introduction Recently, anterior cutaneous nerve entrapment syndrome (ACNES) has been described as a possible cause for chronic abdominal pain in the pediatric and adult population. While the exact incidence of ACNES in the pediatric population remains unknown, ACNES was identified in 13% of children originally diagnosed with functional abdominal pain.[1] Due to the increased awareness, more and more children are being referred to the (pediatric) surgeon
ro of
to evaluate whether or not ACNES might be the cause of the chronic abdominal pain.
The diagnosis of ACNES is based upon patient history and physical examination. Pain is the
-p
most dominant symptom. In addition, coughing, body stretching, or any type of movement or
re
more intense use of abdominal muscles may aggravate the pain.[2] At physical examination,
lP
painful abdominal palpation, positive pinch test and Carnett’s sign may be found.[2,3] The Carnett’s sign helps to distinguish if the pain originates from the abdominal wall or from
na
abdominal viscera. During testing for the Carnett’s sign, the investigator palpates the point of
ur
maximum pain, after which the patient is asked to tense abdominal muscles followed again by palpation at the point of maximum pain. If the pain increases during palpation with flexed
Jo
abdominal muscles, the Carnett’s sign is positive indicating the pain originates from the abdominal wall.[4] Furthermore, altered skin sensibility demonstrated by a swab or alcoholsoaked gauze strongly support the diagnosis.[2,3,5] In addition, one can inject a local anesthetic into the trigger point with or without corticosteroids and with or without ultrasound guidance.[2,6–8] Pain relief for several hours or days confirms the diagnosis.[2,5,9] If multiple injections are only successful for a short period of time, a neurectomy should be considered.[2,10,11]
Journal Pre-proof However, the exact pathophysiology of ACNES is unknown. It is postulated that the abdominal pain is caused by compression or traction of cutaneous nerve branches of intercostal nerves (Th7-Th12).[2,6,12] Although other authors hypothesized that the neuralgia might be caused secondary to respiratory infections or subclinical infection with herpes zoster.[2,4,13] Therefore, the goal of this present study was to determine if nerves resected from children with ACNES show any signs of inflammation or infection, and
ro of
thereby supporting the role of inflammation in the pathogenesis of ACNES.
1. Methods
-p
1.1 Setting
re
In compliance with local ethical board guidelines for clinical studies, we performed a pilot prospective cohort study at the department of pediatric surgery of Emma Children’s Hospital.
lP
Emma Children’s Hospital is a tertiary referral center, is an expert center for children with
na
chronic abdominal pain, and participates in the national network of surgeons treating patients with ACNES. Our team consist of three pediatric surgeons. The surgery was performed by at
Jo
ur
least two surgeons of this team.
1.2 Population and design
Children younger than 18 years who were diagnosed and treated between December 2017 and June 2018 for ACNES by one of the three pediatric surgeons from our ACNES team were eligible for inclusoin. Diagnosis of ACNES was based upon the following predefined criteria: 1. Medical history 2. Physical examination: positive Carnett’s sign 3. Positive reaction on a diagnostic injection with a local anesthetic agent.
Journal Pre-proof If a patient fulfilled the diagnostic criteria, they were treated for ACNES according to our local protocol which consists of a free-hand injection with a local anesthetic (i.e. lidocaine or bupivacaine), but without corticosteroids into the trigger point, up to a maximum of 3 times. If pain persisted after these injections, an anterior neurectomy was performed. Only those patients that underwent an anterior neurectomy in the aforementioned time period were included in this study. Patients were excluded if no nerve tissue was present during
ro of
histopathological examination.
1.3 Surgical procedure
-p
The procedures were performed in a day care surgery setting by a team of two of three
re
surgeons of the ACNES team. Preoperatively, the area of maximal pain was identified and marked (together with the patient) prior to the general anesthesia. The anterior sheath of the
lP
abdominal rectus muscle was exposed via a transverse 5 cm skin incision. Dissection of the
na
subcutaneous fat tissue and Scarpa’s fascia was done with a combination of blunt and cautery dissection. All neurovascular bundles penetrating the fascial foramina of the anterior sheath
ur
were identified within a 5 cm radius. The fascial foramina were widened and the
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neurovascular bundles were resected beyond the fascia after injection of a local anesthetic agent. Subsequently, the resected were sent for routine histopathological examination. All specimens sent for histopathological examination in this time period underwent standard protocol analysis including immunohistochemical staining. Nerves were pinned and marked (skin side, fascia penetration and underneath fascia site). Fresh tissue was directly sent to the department of pathology for fixation.
1.4 Fixation and slides
Journal Pre-proof The tissue samples were fixed overnight in a 4 % formaldehyde and routinely processed into liquid paraffin. Sections were cut in 4-6 µm with a microtome (Microm, Heidelberg, Germany), and mounted on positively charged slides (Superfrost + Menzel, Germany). Each specimen was histopathologically examined using haematoxylin & eosin (H & E) and luxol fast blue (LFB). An immunohistochemical examination of all surgical specimens was performed using the following panel of antibodies: S100 (1:4000, clone Z0311, DAKO, Glostrup, Denmark, pan neural marker), anti-CD68 (mouse monoclonal, clone PG-M1;
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DAKO; 1:200, monocytes, macrophages, microglia) and anti-CD3 (mouse monoclonal, clone F7.2.38; DAKO; 1:200, T lymphocytes). The slides were air dried overnight at 37°C. All
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immunohistochemical stainings were performed with a Ventana semiautomated staining
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machine (Benchmark ULTRA; Ventana, Illkirch, France) and the Ventana DAB staining
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system according to the manufacturer’s protocol.
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1.5 Outcome measures 1.5.1 Primary outcome
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Our primary outcome is defined as the number of patients with signs suggestive for
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inflammation or infection defined as: -
Positive H & E staining: influx of lymphocytes or neutrophils
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Immunohistochemistry: influx of CD3-positive T-lymphocytes
The histopathological assessment was performed by two neuropathologists, who determined if there were any signs of inflammation or infection.
1.5.2. Secondary outcome Our secondary outcomes were treatment success, defined as less pain or no pain after neurectomy, and complication rate during and after surgery.
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1.6 Statistical methods Due to the nature of this study, only descriptive analysis was performed. Primary and secondary outcomes were reported as n (%).
2. Results 2.1 General characteristics
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In this time period, a total of eight children underwent anterior neurectomy, although one patients was excluded from this study due to the fact that on histopathological examination
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no nerve tissue could be identified. General characteristics of the seven included patients are
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shown in table 1, with a median (range) age of 15 (14-16) years old. Six patients (86%) were female, and one patient (14%) had a medical history of abdominal surgery. None of the
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duration was 47 (19-280) days.
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patients showed any signs of skin disease or infection. The median (range) follow-up
2.2 Primary outcome
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2.2.1 Histopathological examination
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All histopathological specimens showed non-specific nerve degeneration. H&E staining did not show any signs of infection or inflammation (see table 2) (see figure 1).
2.2.2 Immunohistochemistry All histopathological specimens were S100 positive, confirming the presence of nerve tissue. There were several CD68-positive macrophages present in the specimens. Four out of seven (57%) specimens showed the presence of a few CD3-positive T-cells, however, this was not suggestive for inflammation (see table 2) (see figure 1).
Journal Pre-proof 2.3 Secondary outcome All seven patients (100%) were pain free after the neurectomy during the follow-up period.
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There were no peri- and postoperative complications.
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3. Discussion This study shows that there is no influx of CD3-positive cells (lymphocytes) and only limited influx of CD68-positive cells (macrophages) in the resected nerves of children undergoing a neurectomy for ACNES. These findings do not support the hypothesis that inflammation or infection might play a role in the pathogenesis of ACNES as postulated in the past by some authors. They stated that the neuralgia in patients with ACNES might be the result of
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secondary inflammation of the nerves due to viral infections.[2,4,13] Results, however, should be interpreted with care as this was a pilot study with only a limited number of
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patients.
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Varicella zoster virus (VZV), an alpha herpesvirus only found in humans, causes varicella
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(chickenpox) upon primary infection.[14–16] After the infection resolves, VZV becomes latent in peripheral nervous system (PNS) ganglia of nearly all previously infected
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individuals with no corresponding morphological changes.[16] After reactivation from a
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latent state it causes herpes zoster (HZ).[14–16] It has been shown that some neurons do not survive after an episode of HZ and that axonal injury, demyelination and inflammation occur
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from the dorsal root to the skin.[17,18] After HZ resolves, many patients experience severe persistent pain (postherpetic neuralgia; PHN), which can persist for months up to years.[14– 16,19] Postmortem microscopic analysis of ganglia in patients with known HZ infection showed the presence of inflammatory cells around neurons.[16,20,21] Since none of the patients showed any signs of skin infection, no signs of infection or inflammation was found during microscopic evaluation and no large presence of CD68-positive histiocytes or influx of CD3-positive T-cells, it is unlikely that inflammation or infection of the nerves plays an important role in ACNES.
Journal Pre-proof Our findings support the hypothesis that ACNES is caused by compression or traction of cutaneous nerve branches of intercostal nerves (Th7-Th12), or by herniation of connective fat tissue surrounding the nerve within the neurovascular channel in the rectus muscle leading to nerve ischemia and pain.[2,6,12] This is reflected by the limited influx of CD68-positive cells with concomitant nonspecific degenerative signs that are also present in other known nerve compression syndromes, of which carpal tunnel syndrome (CTS) is the most common. CTS is caused by traction and compression of the median nerve, causing disorders of the
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intraneural microcirculation, resulting in nerve edema, demyelination and degeneration from the site of compression and beyond.[22] Another nerve compression syndrome is meralgia
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paresthetica. Meralgia paresthetica is caused by compression of the lateral femoral cutaneous
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nerve[23], and causes similar symptoms as ACNES, including pain and numbness.[24] Histopathological evaluation of resected nerves shows reduced fiber density with multifocal
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fiber loss, loss of myelinated fibers, perineurial thickening, Renaut bodies and subperineural
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edema.[25] These findings have also been shown in other nerve compression syndromes, including human ulnar nerves at sites of compression.[26] Accordingly, we found non-
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specific degeneration of nerve fibers, indicated by the CD68-positive histiocytes within the
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nerve bundles, supporting the fact that the degenerative changes are likely to be caused by compression or traction.
To our knowledge, this is the first study reporting the histopathological findings of nerves resected from children diagnosed with ACNES. Still, several limitations are present. One potential limitation is the limited number of patients included in this study. Although it is possible that this might reduce the viability of our conclusion, we still could not find any suggestion of inflammation in eight consecutive patients. Secondly, it remains unclear whether or not the injection with a local anesthetic agent could influence our results. In our
Journal Pre-proof opinion, injection with a local anesthetic could not be of any influence on the degree of inflammation, although it might cause non-specific degenerative changes. Since we did not use corticosteroids in our injection, it is highly unlikely that the injection had any effect on inflammation. Thirdly, there is the possibility of a sampling error. However, the painful area was marked together with the patient before the operation and all nerves encountered in this area were sent for histopathological examination. We therefore believe that the resected
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nerves were representative for the pediatric population.
4. Conclusion
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Our study supports the hypothesis that ACNES is caused by compression of the nerves rather
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than inflammation or infection.
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Acknowledgements
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There are no acknowledgements. This research did not receive any specific grant from
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funding agencies in the public, commercial, or not-for-profit sectors.
Journal Pre-proof Figure Legend Figure 1. Characteristic findings in nerve biopsies from patients with ACNES who underwent neurectomy. (a) H&E staining shows nerve fiber (pink) surrounded by adipose tissue (white) and no influx of either neutrophils or lymphocytes as sign of infection or inflammation. (b) S100 staining confirms the presence of neural tissue. (c) CD68 staining shows the presence of several macrophages (brown cells). (d) CD3 staining
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shows the presence of a single T-lymphocyte (indicated by the arrow).
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References [1] Siawash M, De Jager-Kievit JWA, Ten WTA, Roumen RM, Scheltinga MR. Prevalence of anterior cutaneous nerve entrapment syndrome in a pediatric population with chronic abdominal pain. J Pediatr Gastroenterol Nutr 2016;62:399–402. [2] Scheltinga MR, Roumen RM. Anterior cutaneous nerve entrapment syndrome (ACNES). Hernia 2018;22:507–16. [3] Siawash M, Roumen R, Ten WTA, van Heurn E, Scheltinga M. Diagnostic characteristics of anterior cutaneous nerve entrapment syndrome in childhood. Eur J Pediatr 2018;177:835–9. [4] Carnett JB. Intercostal neuralgia as a cause of abdominal pain and tenderness. Surg Gynecol Obstet 1926;42:625–32. [5] Boelens OB, Scheltinga MR, Houterman S, Roumen RM. Management of anterior cutaneous nerve entrapment syndrome in a cohort of 139 patients. Ann Surg 2011;254:1054–8. [6] Kifer T, Mišak Z, Jadrešin O, Hojsak I. Anterior cutaneous nerve entrapment syndrome in children: A prospective observational study. Clin J Pain 2018;34:670–3. [7] Siawash M, Mol F, Tjon-A-Ten W, Perquin C, van Eerten P, van Heurn E, et al. Anterior rectus sheath blocks in children with abdominal wall pain due to anterior cutaneous nerve entrapment syndrome: a prospective case series of 85 children. Paediatr Anaesth 2017;27:545–50. [8] Sahoo RK, Nair AS. Ultrasound Guided Transversus Abdominis Plane Block for Anterior Cutaneous Nerve Entrapment Syndrome. Korean J Pain 2015;28:284–6. [9] Boelens OBA, Scheltinga MR, Houterman S, Roumen RM. Randomized clinical trial of trigger point infiltration with lidocaine to diagnose anterior cutaneous nerve entrapment syndrome. Br J Surg 2013;100:217–21. [10] Siawash M, Maatman R, Tjon A. Ten W, van Heurn E, Roumen R, Scheltinga M. Anterior neurectomy in children with a recalcitrant anterior cutaneous nerve entrapment syndrome is safe and successful. J Pediatr Surg 2017;52:478–80. [11] Armstrong LB, Dinakar P, Mooney DP. Neurectomy for anterior cutaneous nerve entrapment syndrome in children. J Pediatr Surg 2018;53:1547–9. [12] Akhnikh S, De Korte N, De Winter P. Anterior cutaneous nerve entrapment syndrome (ACNES): The forgotten diagnosis. Eur J Pediatr 2014;173:445–9. [13] Davis JH. Segmental neuralgia in childhood simulating visceral disease. JAMA 1936;107:1620–6. [14] Kinchington PR, Goins WF. Varicella zoster virus-induced pain and post-herpetic neuralgia in the human host and in rodent animal models. J Neurovirol 2011;17:590–9. [15] Gilden D, Nagel M, Cohrs R, Mahalingam R, Baird N. Varicella Zoster Virus in the Nervous System. F1000Research 2015;4. doi:10.12688/f1000research.7153.1. [16] Kleinschmidt-DeMasters BK, Gilden DH. Varicella-zoster virus infections of the nervous system: Clinical and pathologic correlates. Arch Pathol Lab Med 2001;125:770–80. [17] Petersen KL, Rice FL, Farhadi M, Reda H, Rowbotham MC. Natural history of cutaneous innervation following herpes zoster. Pain 2010;150:75–82. [18] Watson C, Deck JH. The neuropathology of herpes zoster with particular reference to postherpetic neuralgia and its pathogenesis. In: Watson C, editor. Herpes zoster and postherpetic neuralgia, Amsterdam: Elsevier Inc.; 1993, p. 139–58. [19] Gilden DH, Kleinschmidt-DeMasters BK, LaGuardia JJ, Mahalingam R, Cohrs RJ. Neurologic Complications of the Reactivation of Varicella–Zoster Virus. N Engl J Med 2000;342:635–45. [20] Watson CPN, Deck JH, Morshead C, Van der Kooy D, Evans RJ. Post-herpetic
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[26]
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[24]
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[21]
neuralgia: Further post-mortem studies of cases with and without pain. Pain 1991;44:105–17. Smith FP. Pathological studies of spinal nerve ganglia in relation to intractable intercostal pain. Surg Neurol 1978;10:50–3. Aboonq MS. Pathophysiology of carpal tunnel syndrome. Neurosciences (Riyadh) 2015;20:4–9. de Ruiter GCW, Lim J, Thomassen BJW, van Duinen SG. Histopathologic changes inside the lateral femoral cutaneous nerve obtained from patients with persistent symptoms of meralgia paresthetica. Acta Neurochir (Wien) 2019;161:263–9. Cheatham SW, Kolber MJ, Salamh PA. Meralgia paresthetica: a review of the literature. Int J Sports Phys Ther 2013;8:883–93. Berini SE, Spinner RJ, Jentoft ME, Engelstad JK, Staff NP, Suanprasert N, et al. Chronic meralgia paresthetica and neurectomy: a clinical pathologic study. Neurology 2014;82:1551–5. Neary D, Eames RA. The pathology of ulnar compression in man. Neuropathol Appl Neurobiol 1975;1:69.
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Table 1. Patient characteristics General characteristics Age (years) 15 (14-16) Female. No (%) 6 (86) Previous abdominal surgery. 1 (14) No (%) Skin disease or infection 0 (0) during physical examination. No (%) Data displayed as median (range), unless stated otherwise
Journal Pre-proof Table 2. Histopathological changes in nerves of children with ACNES Nerve tissue
CD68positive
CD3positive
Microscopic histopathological evaluation Fibro-adipose tissue with nerve fibers showing degenerative changes. No inflammation. Nerve tissue with degenerative changes. No inflammation.
1
Yes
Yes
No
2
Yes
Yes
Yes
3
Yes
Yes
Yes
Fibro-adipose tissue with nerve fibers. No inflammation. Vascular structures show hemorrhagic changes.
4
Yes
Yes
No
5
Yes
Yes
No
6
Yes
Yes
Yes
Fibro-adipose tissue with nerve fibers showing degenerative changes. No inflammation. Fibro-adipose tissue with nerve fibers showing degenerative changes. No inflammation. Fibro-adipose tissue with nerve fibers showing degenerative changes. No inflammation.
7
Yes
Yes
Yes
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Fibro-adipose tissue with nerve fibers showing degenerative changes. No inflammation.
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Immunohistochemistry
Conclusion
CD68 shows the presence a few macrophages.
No signs of inflammation
CD68 shows the presence of macrophages in the nerve fibers. A few CD3-positive T-cells are present, without aggregation. CD68 shows the presence of macrophages in the nerve fibers. A few CD3-positive T-cells are present, however, not around the nerve fibers. CD68 shows the presence of a few macrophages in the nerve fibers. CD68 shows the presence of a few macrophages around the nerve fibers. CD68 shows the presence of a few macrophages around the nerve fibers. A few CD3-positive T-cells are present. CD68 shows the presence of a few macrophages around the nerve fibers. A few CD3-positive T-cells are present.
No signs of inflammation
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Patient
No signs of inflammation
No signs of inflammation
No signs of inflammation
No signs of inflammation
No signs of inflammation
Figure 1