Techniques for the standard histological and ultrastructural assessment of nerve biopsies.

It is always a challenge to acquire a clear picture of the pathological processes and changes in any disease. For this purpose, it is advantageous to directly examine the affected organ. Nerve biopsy has been a method of choice for decades to classify peripheral neuropathies and to find clues to uncover their etiology. The histologic examination of the peripheral nerve provides information on axonal or myelin pathology as well as on the surrounding connective tissue and vascularization of the nerve. Minimal requirements of the workup include paraffin histology as well as resin semithin section histology. Cryostat sections, teased fiber preparations and electron microscopy are potentially useful in a subset of cases. Here we describe our standard procedures for the workup of the tissue sample and provide examples of diagnostically relevant findings.

Quantitative assessment of intraneural vascular alterations in peripheral nerve trauma using high-resolution neurosonography: technical note.

High-resolution neurosonography (HRNS) has become a major imaging modality in assessment of peripheral nerve trauma in the recent years. However, the vascular changes of traumatic lesions have not been quantitatively assessed in HRNS. Here, we describe the vascular-ratio, a novel HRNS-based quantitative parameter for the assessment of intraneural vascular alterations in patients with nerve lesions. N = 9 patients suffering from peripheral nerve trauma were examined clinically, electrophysiologically and with HRNS (SonoSite Exporte, Fuji). Image analyses using Fiji included determination of the established fascicular ratio (FR), the cross-section ratio (CSR), and as an extension, the calculation of a vascular ratio (VR) of the healthy versus damaged nerve and a muscle perfusion ratio (MPR) in comparison to a healthy control group. The mean VR in the healthy part of the affected nerve (14.14%) differed significantly (p < 0.0001) from the damaged part (VR of 43.26%). This coincides with significant differences in the FR and CSR calculated for the damaged part versus the healthy part and the controls. In comparison, there was no difference between VRs determined for the healthy part of the affected nerve and the healthy controls (14.14% / 17.72%). However, the MPR of denervated muscles was significantly decreased compared to the non-affected contralateral controls. VR and MPR serve as additional tools in assessing peripheral nerve trauma. Image analysis and calculation are feasible. Combined with the more morphologic FR and CSR, the VR and MPR provide a more detailed insight into alterations accompanying nerve trauma.

New method for the assessment of perineural invasion from perihilar cholangiocarcinoma.

PURPOSE: Perineural invasion (PN) is often found in perihilar cholangiocarcinoma. New procedure was developed to assess PN around the right hepatic artery (RHA) using dual-energy computed tomography (DECT). METHODS: Thirty patients with perihilar cholangiocarcinoma who underwent DECT before biliary drainage were retrospectively reviewed. Mask images, i.e., the periarterial layer (PAL) around the RHA and superior mesenteric artery (SMA), were made from late arterial phase DECT. The mean CT number of the PAL was measured. RESULTS: Twenty patients with PN around the RHA were classified into the PN (+) group. The remaining 10 patients without PN and other 26 patients with other diseases that are never accompanied with PN were classified into the PN (-) group. The PAL ratio (the CT number of the PAL around the RHA relative to that around the SMA) was calculated. Both the mean CT number of the PAL around the RHA and the PAL ratio were significantly higher in the PN (+) group than in the PN (-) group. According to an ROC analysis, the predictive ability of the PAL ratio was superior. Using the cutoff value of the PAL ratio 1.009, a diagnosis of PN around the RHA was made with approximately 75% accuracy. CONCLUSIONS: Assessment with CT number of the PAL reconstructed from DECT images is an easy and objective method to diagnose PN.

A novel endpoint for the assessment of chemotherapy-induced peripheral neuropathy in rodents: biomechanical properties of peripheral nerve.

Chemotherapy-induced peripheral neuropathy (CiPN) is a frequent adverse effect in patients and a leading safety consideration in oncology drug development. Although behavioral assessment and microscopic examination of the nerves and dorsal root ganglia can be incorporated into toxicity studies to assess CiPN risk, more sensitive and less labor-intensive endpoints are often lacking. In this study, rats and mice administered vincristine (75 µg kg-1  day-1 , i.p., for 10 days in rats and 100 µg kg-1  day-1 , i.p., for 11 days in mice, respectively) were employed as the CiPN models. Behavioral changes were assessed during the dosing phase. At necropsy, the sural or sciatic nerve was harvested from the rats and mice, respectively, and assessed for mechanical and histopathological endpoints. It was found that the maximal load and the load/extension ratio were significantly decreased in the nerves collected from the animals dosed with vincristine compared with the vehicle-treated animals (P < 0.05). Additionally, the gait analysis revealed that the paw print areas were significantly increased in mice (P < 0.01), but not in rats following vincristine administration. Light microscopic histopathology of the nerves and dorsal root ganglia were unaffected by vincristine administration. We concluded that ex vivo mechanical properties of the nerves is a sensitive endpoint, providing a new method to predict CiPN in rodent. Gait analysis may also be a useful tool in these pre-clinical animal models.

Geographic tongue: assessment of peripheral nerve status, Langerhans cell, and HLA-DR expression.

OBJECTIVES: The objectives of this study were to determine whether geographic tongue (GT) is an antigen-driven condition by assessing Langerhans cell numbers and the expression of human leukocyte antigen (HLA)-DP, -DQ, and -DR in the epithelium of GT and to assess peripheral nerve status for any possible damage/injury association by quantifying neurite area in connective tissue in GT. STUDY DESIGN: Randomly selected samples of GT were examined by using routine immunoperoxidase staining methods to S100 protein, neurofilament, CD1a, and HLA class II. The Student t test and Mann-Whitney U test were used to assess statistical significance. RESULTS: Langerhans cell numbers were found to be increased in GT. HLA expression was also seen in Langerhans cells and inflammatory cells and in the spinous layer and parabasal epithelial cells in 2 samples of GT. Total nerve tissue, based on area measurements, was not significantly different between GT and control tissues. CONCLUSIONS: The increase in Langerhans cells suggests that GT is a condition that is likely driven by an unknown external antigen. Peripheral nerve damage was not apparent, suggesting that this is not a mechanism whereby patients with GT become symptomatic.

The utility of ultrasound in the assessment of traumatic peripheral nerve lesions: report of 4 cases.

Ultrasound technology continues to improve with better image resolution and availability. Its use in evaluating peripheral nerve lesions is increasing. The current review focuses on the utility of ultrasound in traumatic injuries. In this report, the authors present 4 illustrative cases in which high-resolution ultrasound dramatically enhanced the anatomical understanding and surgical planning of traumatic peripheral nerve lesions. Cases include a lacerating injury of the sciatic nerve at the popliteal fossa, a femoral nerve injury from a pseudoaneurysm, an ulnar nerve neuroma after attempted repair with a conduit, and, finally, a spinal accessory nerve injury after biopsy of a supraclavicular fossa lesion. Preoperative ultrasound images and intraoperative pictures are presented with a focus on how ultrasound aided with surgical decision making. These cases are set into context with a review of the literature on peripheral nerve ultrasound and a comparison between ultrasound and MRI modalities.

Controversies in the pathological assessment of colorectal cancer.

Pathologic assessment of colorectal cancer specimens plays an essential role in patient management, informing prognosis and contributing to therapeutic decision making. The tumor-node-metastasis (TNM) staging system is a key component of the colorectal cancer pathology report and provides important prognostic information. However there is significant variation in outcome of patients within the same tumor stage. Many other histological features such as tumor budding, vascular invasion, perineural invasion, tumor grade and rectal tumor regression grade that may be of prognostic value are not part of TNM staging. Assessment of extramural tumor deposits and peritoneal involvement contributes to TNM staging but there are some difficulties with the definition of both of these features. Controversies in colorectal cancer pathology reporting include the subjective nature of some of the elements assessed, poor reporting rates and reproducibility and the need for standardized examination protocols and reporting. Molecular pathology is becoming increasingly important in prognostication and prediction of response to targeted therapies but accurate morphology still has a key role to play in colorectal cancer pathology reporting.

Histological assessment of thermal damage in the brain following infrared neural stimulation.

BACKGROUND: Infrared neural stimulation (INS) is a novel technique for modulating neural function. Its advantages over electrical stimulation include high spatial specificity, lack of electrical artifact and contact-free stimulation. INS acts via a rapid, focal increase in temperature. However, in order to become a viable experimental and therapeutic tool, the safety of INS must be demonstrated. OBJECTIVE/HYPOTHESIS: Our aim was to determine the upper limit for the radiant exposure of INS in the brain without causing damage, using an INS sequence previously shown to induce both behavioral and electrophysiological effects in rodents and non-human primates. METHODS: We stimulated the brains of anesthetized rodents and two squirrel monkeys using an infrared laser, depositing radiant energies from 0.3 to 0.9 J/cm2 per pulse in 0.5 s-long 200 Hz trains. At the end of the experiment, the animals were euthanized, perfused and the brains processed using standard histological techniques. RESULTS: Radiant exposures greater than or equal to 0.4 J/cm2 resulted in identifiable lesions in brain sections. The lesions had a shape of a parabola and could further be subdivided into three concentric zones based on the type of damage observed. CONCLUSIONS: The thermal damage threshold following our INS paradigm was between 0.3 and 0.4 J/cm2 per pulse. This value is lower than the one found previously in peripheral nerve. The differences are likely due to the structure of the INS sequence itself, particularly the repetition rate. The results warrant further modeling and experimental work in order to delimit the INS parameter space that is both safe and effective.

Prognostication of vulvar cancer based on p14ARF status: molecular assessment of transcript and protein.

PURPOSE: This study was designed to determine the prognostic role of p14ARF in vulvar squamous cell carcinoma (VSCC). METHODS: Immunohistochemistry for p14ARF and p53 and fluorescent in situ hybridization (FISH) for TP53 were performed in 139 cases of VSCC. Human papillomavirus (HPV) genotyping by hybridization was employed in 100 cases. qRT-PCR for p14ARF and p53 transcript assessment was performed in 16 cases. All results were correlated with clinicopathological variables. RESULTS: Immunohistochemistry analysis showed p14ARF and p53 positivity in 16.4% and 53% cases respectively. Positive p14ARF expression was significantly associated with the following variables: shorter cancer-specific survival (P=0.04) and shorter disease-free survival (P=0.02), presence of perineural invasion (P=0.037), vascular invasion (P=0.047), and node metastasis (P=0.031). Also, p14ARF-positive HPV-negative cases had the shortest cancer-specific survival (P=0.03) and disease-free survival (P=0.04). HPV infection was detected in 32.8% of the cases; HPV16 was the most prevalent type. Viral infection was more common in poorly differentiated tumors (P=0.032). qRT-PCR demonstrated that CDKN2A (p14ARF) had higher expression in tumor samples compared with paired noncancerous samples (P<0.001). The opposite relationship was seen in TP53 expression evaluation (P<0.001). FISH demonstrated 4 cases with deleted TP53 (6.3%). CONCLUSIONS: p14ARF represents an important marker of poor prognosis in VSCC. p53 and HPV infection did not show any prognostic importance. Further clinical trials concerning p14ARF positivity may result in important contributions due to its relationship with poor outcome. Mainly due to the relationship of p14ARF with lymph node metastasis, the immunohistochemistry evaluation of this marker may help to identify a subset of patients more suitable to less radical procedures.

Musculoskeletal ultrasound for peripheral nerve lesions.

Although the combination of a detailed physical examination and a subsequent electrodiagnostic study is used for the diagnosis of peripheral nerve disorders, prompt imaging may also be necessary in daily practice. In this regard, as having higher spatial resolution, and being a faster, more cost-effective and dynamic study; ultrasound (US) has become a very convenient first-line imaging modality for the diagnosis, follow-up and treatment (i.e. guiding interventions or planning for surgery) of peripheral nerve pathologies. Yet, using the probe of US to "sono-auscultate" the peripheral nerves is indisputably paramount for unmasking the whole scenario of injury. Likewise, in this review, we will try to exemplify the role of US for the diagnosis and follow-up of peripheral nerve disorders in clinical practice.

Difficulties with the neurological assessment of humans following a chimpanzee attack.

Chimpanzee attacks can be vicious, mutilating, and disabling if not fatal. Stereotypically, the hands and face are targeted, and in male victims, genitalia are mutilated. The authors present a case highlighting the difficulties with early neurological assessment following such an attack. This 55-year-old woman was attacked by a 14-year-old chimpanzee. She suffered mutilation of both hands, severe midface bony, soft-tissue, and eye injuries, and scalp degloving. An emergency tracheotomy was performed at the scene, with an unclear duration of hypoxia. The patient was unresponsive without spontaneous movements, papillary or corneal reflexes, cough, or gag. Attempts to lighten sedation were not tolerated. Brain CTs were normal. Intracranial pressure monitoring was deemed infeasible. Brain MR imaging suggested diffuse axonal injury consistent with severe shaking trauma. Diffusion tensor imaging indicated intact corticospinal tracts, confirmed by somatosensory evoked potentials. Magnetic resonance imaging suggested left optic nerve transaction, and right retinal detachment was diagnosed. Electroencephalography showed severe diffuse encephalopathy. Auditory evoked potentials showed absent auditory pathway responses except for a right delayed wave V. Visual evoked potentials indicated absent visual function. At 1 month after the attack, sedation and analgesia weaning revealed lower-extremity movement to command, but no upper-limb response. Cervical spine and brachial plexus MR imaging showed brachial plexus edema. Two months after the attack, the patient regained strength in all her extremities and verbally communicated using a Passy-Muir tracheostomy valve. Chimpanzee attacks on humans can cause extensive, life-threatening injuries. The neurological assessment of such patients is challenging, complicated by limb and craniofacial disfigurement and the need for heavy sedation. Initial assessment of nervous system integrity may rely on costly imaging and electrophysiological studies.

Correlation and dissociation of electrophysiology and histopathology in the assessment of toxic neuropathy.

The evaluation of neurotoxic damage involves a unique set of challenges. Vulnerable structures, such as neocortex, hippocampus, spinal cord, and peripheral nerve are complex and sharply differentiated; deficits can result from insults to one or more element(s) in the system (e.g., myelin, axon, soma, synapse, or glia). In-life assessment of neurotoxic damage is complicated by the relative inaccessibility of structures in the brain and spinal cord, and recovery is severely limited. Histopathology and electrophysiology represent two of the most commonly used and valuable techniques in this field. This review outlines the strengths and limitations of these procedures and focuses on circumstances in which findings from these measures are dissociated. Electrophysiology is noninvasive and affords a longitudinal view of onset and progression of deficits; however, measures are generally weighted to large-diameter myelinated axons and to regions of primary sensory and motor processing. Histology is a highly validated biomarker, but it is restricted by sampling issues and is insensitive to some elements of neurotoxicity (e.g., altered channel function) associated with profound functional consequences. The central tenet of the discussion is that histology and electrophysiology offer complementary views of neurotoxic damage and, whenever possible, they should be used in concert.

Traumatic neuropathies: spectrum of imaging findings and postoperative assessment.

Traumatic injury to peripheral nerves is a significant cause of morbidity and disability. Until reinnervation occurs, electrodiagnostic studies cannot differentiate severe axonotmetic lesions (Sunderland class 4) from complete nerve transection or neurotmesis (Sunderland class 5). This limitation is relevant clinically because in cases of neurotmesis an improved outcome may be achieved with an early surgical repair (within 1 week after trauma). High-resolution ultrasound (US) is an efficient modality to visualize injured nerves and is becoming increasingly important among radiologists and surgeons. Magnetic resonance (MR) imaging is complementary to high-resolution US, especially in evaluating deep-seated and proximal nerve segments. This article describes the imaging features of traumatic peripheral nerve lesions. The role of diagnostic imaging in stretching injuries, contusion trauma, penetrating wounds, and after surgery is discussed. A multimodality diagnostic approach including physical examination, electrophysiology, and US and MR imaging allows an accurate evaluation of most peripheral nerves. Imaging assessment of peripheral nerves trauma is useful for the diagnosis, follow-up, and postoperative evaluation.

Electrodiagnostic assessment of peripheral neuropathies.

The authors discuss the techniques and use of electrodiagnosis to help fully characterize peripheral neuropathies, including electrodiagnostic principles, normal findings, correlations between underlying nerve pathology that leads to abnormal electrodiagnostic findings, and how to detect and interpret electrodiagnostic findings.

Assessment of perineural infiltration and spread of oral squamous cell carcinoma: a clinicohistopathologic study.

BACKGROUND: As the tumor spreads through the pathway of least resistance, the present study was carried out to evaluate the presence of perineural infiltration and spread of oral squamous cell carcinoma (OSCC) along the perineural spaces in gingivobuccal sulcus tumors infiltrating into the mandible. AIMS AND OBJECTIVES: (1) To investigate the incidence of perineural invasion of OSCC along the inferior alveolar nerve and (2) to investigate the neurovascular bundle as a potential route of spread of OSCC. MATERIALS AND METHODS: Twenty-six patients with histopathologically proven OSCC of the gingivobuccal sulcus with radiographic infiltration of the mandible were included. The surgical specimens were decalcified and serially sectioned. Each section was stained with hematoxylin and eosin and was screened for the presence of perineural invasion and spread. Results : Twenty-five specimens showed perineural infiltration but none of the cases showed perineural spread along the inferior alveolar canal. Also, not all cases showed any neurologic deficit. Follow-up of these cases showed early recurrence (6-8 months) in the study group. CONCLUSION: Perineural infiltration is present in OSCC but perineural spread along the inferior alveolar canal is absent. It is a bad prognostic indicator.

Magnetic resonance neurography and diffusion tensor imaging: origins, history, and clinical impact of the first 50,000 cases with an assessment of efficacy and utility in a prospective 5000-patient study group.

OBJECTIVE: Methods were invented that made it possible to image peripheral nerves in the body and to image neural tracts in the brain. The history, physical basis, and dyadic tensor concept underlying the methods are reviewed. Over a 15-year period, these techniques-magnetic resonance neurography (MRN) and diffusion tensor imaging-were deployed in the clinical and research community in more than 2500 published research reports and applied to approximately 50,000 patients. Within this group, approximately 5000 patients having MRN were carefully tracked on a prospective basis. METHODS: A uniform Neurography imaging methodology was applied in the study group, and all images were reviewed and registered by referral source, clinical indication, efficacy of imaging, and quality. Various classes of image findings were identified and subjected to a variety of small targeted prospective outcome studies. Those findings demonstrated to be clinically significant were then tracked in the larger clinical volume data set. RESULTS: MRN demonstrates mechanical distortion of nerves, hyperintensity consistent with nerve irritation, nerve swelling, discontinuity, relations of nerves to masses, and image features revealing distortion of nerves at entrapment points. These findings are often clinically relevant and warrant full consideration in the diagnostic process. They result in specific pathological diagnoses that are comparable to electrodiagnostic testing in clinical efficacy. A review of clinical outcome studies with diffusion tensor imaging also shows convincing utility. CONCLUSION: MRN and diffusion tensor imaging neural tract imaging have been validated as indispensable clinical diagnostic methods that provide reliable anatomic pathological information. There is no alternative diagnostic method in many situations. With the elapsing of 15 years, tens of thousands of imaging studies, and thousands of publications, these methods should no longer be considered experimental.

[Neurolymphomatosis: diagnosis of extension and assessment of response to treatment with PET-CT]. / Neurolinfomatosis: diagnóstico de extensión y valoración de la respuesta al tratamiento mediante PET-TAC.

Neurolymphomatosis is a rare neurological manifestation of non-Hodgkin's lymphoma (NHL) and it may be its first and sole manifestation. Diagnosis is often difficult and nerve biopsy is generally required. However, this it is not always possible to perform or is not conclusive. We present the case of a 66-year-old woman diagnosed with giant B-cell NHL. After 6 cycles of chemotherapy, imaging and molecular biology techniques showed complete remission. At four months after treatment, the patient complained of low back pain of radicular distribution. CT and MRI imaging showed signs of lymphoproliferative activity of L5 and also lesions to thoracic nerve roots. A PET-CT was requested in order to complete the diagnosis and plan the treatment. Imaging confirmed the presence of tumor recurrence with neurolymphomatosis and also indicated lesions on the chest and abdominal level. Thus, it was decided to start a new line of chemotherapy, without performing the histological study through biopsy. This case illustrates the important role played by PET-CT imaging in neurolymphomatosis diagnosis. This technique can help the patient avoid more aggressive procedures, such as a biopsy, and can also be useful in the follow-up and assessment of the treatment response to NHL-diagnosed patients.

The value of skin biopsy with recording of intraepidermal nerve fiber density and quantitative sensory testing in the assessment of small fiber involvement in patients with different causes of polyneuropathy.

The primary aim of our study was to demonstrate how the diagnostic characteristics of skin biopsy used to evaluate small fiber involvement in patients with different causes of polyneuropathy are intrinsically related to the method used to establish the reference values (cut-off values). We also investigated intraepidermal nerve fiber (IENF) density and abnormalities in quantitative sensory testing (QST) in patients with different causes of polyneuropathy and signs of small fiber involvement. A total of 210 patients with symptoms and signs of polyneuropathy were entered into the study. All patients underwent neurological examination, nerve conduction studies, QST on the thigh and distal part of the calf with detection of warm and cold perception thresholds, and skin biopsy with assessment of IENF density. Cut-off values for IENF density were established from our reference material using Z-scores (calculated from multiple regression analysis), fifth percentile, and receiver operating characteristic (ROC) analysis. Of the patients participating in the study, 65 had an established diagnosis of diabetes mellitus, 70 were classified with idiopathic polyneuropathy, and 75 had other possible causes of polyneuropathy. Forty-five patients met the criteria for small fiber polyneuropathy (SFN), and the remaining 165 had also involvement of large nerve fibers. Of the total patient cohort, 84 (40%) had reduced IENF density based on the Z-score, and 106 patients (50%) had at least one abnormality based on QST. In the SFN group, skin biopsy showed a sensitivity of 31% and a specificity of 98% when reference values were presented with Z-scores. When the fifth percentile was used as the cut-off value (6.7 fibers/mm), sensitivity was 35% and specificity 95%. Applying the ROC analysis with a chosen sensitivity of 78% and specificity of 64%, we had a cut-off point of 10.3 fibers/mm. We conclude that skin biopsy with assessment of IENF is a useful method for investigating patients with SFN. The diagnostic value of the test, however, depends upon on the approach used to estimate the reference values.

Biopsy of the posterior interosseous nerve: a low morbidity method for assessment of peripheral nerve disorders.

AIMS: The sural nerve is the commonest peripheral nerve biopsied to help in the diagnosis of peripheral neuropathy of unknown cause. However, associated complications limit its use. The aim was, as an alternative, to asses biopsy of the terminal branch of the posterior interosseous nerve (PIN) in the forearm. METHODS: PIN pathology was morphometrically quantified in 10 male patients with Type 2 diabetes and compared with six PIN biopsy specimens taken post mortem from male cadavers with no history of neuropathy or trauma. RESULTS: The PIN biopsy procedure provides a long (approximately 3 cm) mono- or bifascicular nerve biopsy with generous epineurial tissue and adjacent vessels. Our results show a significantly lower myelinated fibre density in subjects with diabetes [5782 (3332-9060)/mm(2)] compared with autopsy control material [9256 (6593-12,935)/mm(2), P < 0.007]. No postoperative discomfort or complications were encountered. CONCLUSIONS: A reduction in myelinated fibre density has previously been shown to be a clinically meaningful measure of neuropathy in diabetic patients. We demonstrate similar findings using the PIN biopsy. The PIN biopsy procedure fulfils the criteria for nerve biopsy and was well tolerated by the patients. It may be a possible alternative to sural nerve biopsy to allow for diagnosis of neuropathy.