Nerve biopsy--some comments on procedures and indications.

Nerve biopsy is most often a final step in the evaluation of patients with peripheral neuropathy. The procedure should always be expected to result in varying degree of sensory loss within the innervation area of the biopsied nerve and chronic pain in the area may also occur. Therefore appropriate informed consent must be obtained and a weighing of such side effects and benefits for the patient, particularly therapeutical consequences, should be seriously considered before the procedure is performed. The surgical procedure and the processing in the laboratory of the nerve material must hold a high standard at all levels. Nerve biopsy should not be performed before adequate clinical, electrophysiological and laboratory investigations have been performed. The choice of nerve is important, but in most instances the sural nerve is biopsied, although the superficial peroneal nerve is also an option and allows an easy access to muscle biopsy in the same procedure. Laboratories performing nerve biopsies should have the facilities and expertise to prepare and evaluate fixed and frozen sections (paraffin, cryostat and epoxy-sections) and teased fibers, and also to perform light and electron microscopy and immunohistochemistry. Although not routinely used, the option of morphometry should be available as well. We recommend that properly trained technicians start the processing procedures in the operating room and, if feasible, even in hospitals outside that of the hospital with nerve laboratory. We also prefer routine use of teased fiber analysis as this visualizes in an excellent way pathological processes like axonal degeneration, demyelination and remyelination as well as other features. Evaluation of small fiber neuropathy is rarely an indication for nerve biopsy and should be investigated with skin biopsy and visualization and quantification of intraepidermal nerve fibers. Investigation of inflammatory neuropathy, particularly to demonstrate nerve vasculitis, is the main indication of nerve biopsy.

Pregnancy protects against antiangiogenic and fibrogenic effects of angiotensin II in rat hearts.

AIM: To investigate the difference between physiological and pathological cardiac remodelling induced, respectively, by pregnancy and angiotensin (Ang) II, and to test the hypothesis that pregnancy protects against Ang II effects. METHODS: Female Wistar rats, pregnant (n = 12) and non-pregnant (n = 12), were implanted with mini-pumps containing saline (sham) or 150 ng kg(-1) min(-1) Ang II. Ten days later echocardiography and blood pressure measurement were performed. Expression of 22 genes was assessed using RT-PCR. Microscopic sections of LV were prepared to determine collagen content (Sirius Red staining), vessel density (ß-actin immunolabelling) and myocytes diameter (Toluidine Blue). RESULTS: Heart weight (HW) was increased in Ang II treated groups compared with their controls. Furthermore, HW of Ang II treated pregnant rats (1.0 ± 0.03 g) was higher than that in non-pregnant sham (0.7 ± 0.02 g), pregnant (0.8 ± 0.01 g) and Ang II treated non-pregnant (0.8 ± 0.02 g) rats. Relative LV wall thickness showed similar pattern. Aortic pressure was significantly increased in Ang II groups. Collagen content was increased in Ang II (4.0 ± 0.5%) compared with sham (1.5 ± 0.1%) but reduced again when treated rats were pregnant (2.8 ± 0.4%). Vessel density was reduced by 47.8% after Ang II treatment in non-pregnant and by only 13.9% in pregnant rats. Gene expression analysis showed increased expression of atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), anykrin repeat domain-containing protein 1 (Ankrd-1), protein kinase C-α and -δ and tumour suppressor gene TP53 (p53) in Ang II treated groups and upregulation of ANF, BNP and Ankrd-1 remained when pregnancy was combined with Ang II. Pregnancy reduced expression of: α-myosin heavy chain, tumour necrosis factor-α, p53, endothelial nitric oxide synthase and inducible nitric oxide synthase. CONCLUSION: Pregnancy seems to counteract the detrimental effects of Ang II on fibrosis and angiogenesis in heart. In addition, pregnancy and Ang II lead to partly opposite changes in the expression of some genes important for heart function.

Gene expression, function and ischemia tolerance in male and female rat hearts after sub-toxic levels of angiotensin II.

To examine the response to chronic high-dose angiotensin II (Ang II) and a proposed milder response in female hearts with respect to gene expression and ischemic injury. Female and male litter-matched rats were treated with 400 ng kg(-1) min(-1) Ang II for 14 days. Hearts were isolated, subjected to 30-min ischemia and 30-min reperfusion in combination with functional monitoring and thereafter harvested for gene expression, WB and histology. Ang II-treated hearts showed signs of non-hypertrophic remodeling and had significantly higher end diastolic pressure after reperfusion, but no significant gender difference was detected. Ang II increased expression of genes related to heart function (ANF, ß-MCH, Ankrd-1, PKC-α, PKC-δ TNF-α); fibrosis (Col I-α1, Col III-α1, Fn-1, Timp1) and apoptosis (P53, Casp-3) without changing heart weight but with 68% increase in collagen content. High (sub-toxic) dose of Ang II resulted in marked heart remodeling and diastolic dysfunction after ischemia without significant myocyte hypertrophy or ventricular chamber dilatation. Although there were some gender-dependent differences in gene expression, female gender did not protect against the overall response.

Altered brain myelin sheath morphology after rewarming in situ.

In this study cerebral ultrastructure was examined in an in vivo rat model, after rewarming from profound hypothermia (15-13 degrees C). Animals held at 37 degrees C served as controls. After rewarming, brains were examined by electron microscope. Micrographs were taken randomly, analyzed anonymously, and quantified by morphometry. Serum analysis of the stress marker S-100beta was carried out in identical groups. The most striking findings in rewarmed animals, when compared to controls, were alterations of myelin sheaths (p<.008) and elevated S-100beta (p<.0001). This indicates that cells in the central nervous system are susceptible to injury in an experimental model of accidental hypothermia and rewarming.

Production of mesons and baryons at high rapidity and high p(T) in proton-proton collisions at square root[s] = 200 GeV.

We present particle spectra for charged hadrons pi(+/-), K(+/-), p, and p[over] from pp collisions at square root[s] = 200 GeV measured for the first time at forward rapidities (2.95 and 3.3). The kinematics of these measurements are skewed in a way that probes the small momentum fraction in one of the protons and large fractions in the other. Large proton to pion ratios are observed at values of transverse momentum that extend up to 4 GeV/c, where protons have momenta up to 35 GeV. Next-to-leading order perturbative QCD calculations describe the production of pions and kaons well at these rapidities, but fail to account for the large proton yields and small p[over]/p ratios.

Intraepidermal nerve fibre density, quantitative sensory testing and nerve conduction studies in a patient material with symptoms and signs of sensory polyneuropathy.

Small diameter nerve fibre (SDNF) neuropathy is an axonal sensory neuropathy affecting unmyelinated (C) and thin myelinated (A-delta) fibres. We have evaluated 75 patients with symptoms and signs suggesting SDNF dysfunction with or without symptoms and signs of co-existing large diameter nerve fibre involvement. The patients were examined clinically and underwent skin biopsy, quantitative sensory testing (QST) and nerve conduction studies (NCS). The purpose of this study was to compare the relationship between the different methods and in particular measurements of thermal thresholds and intraepidermal nerve fibre (IENF) density in the same site of the distal leg. The main subdivision of the patient material was made according to the overall NCS pattern. Patients with normal NCS (38) had 6.4 +/- 3.8 and patients with abnormal NCS (37) had 4.4 +/- 3.4 IENF per mm (P = 0.02). Limen (difference between warm and cold perception thresholds) was significantly higher (more abnormal) in those with abnormal than in those with normal NCS (22.1 +/- 9.1 vs. 13.4 +/- 5.6, P < 0.0001). Cold perception threshold was more abnormal (P < 0.0001) than warm perception threshold (P = 0.002). Correlation between IENF and QST was statistically significant only when NCS was abnormal, and thus dependent of a more severe neuropathic process in SDNFs.

Centrality dependence of charged-particle pseudorapidity distributions from d + Au collisions at sqrt[sNN] = 200 GeV.

Charged-particle pseudorapidity densities are presented for the d + Au reaction at sqrt[s(NN)] = 200 GeV with -4.2 < or = eta < or = 4.2. The results, from the BRAHMS experiment at BNL Relativistic Heavy-Ion Collider, are shown for minimum-bias events and 0%-30%, 30%-60%, and 60%-80% centrality classes. Models incorporating both soft physics and hard, perturbative QCD-based scattering physics agree well with the experimental results. The data do not support predictions based on strong-coupling, semiclassical QCD. In the deuteron-fragmentation region the central 200 GeV data show behavior similar to full-overlap d+Au results at sqrt[s(NN)] = 19.4 GeV.

Morphologic changes in tubular cells from in situ kidneys following experimental hypothermia and rewarming.

Although renal failure may occur following rewarming from deep accidental hypothermia, this subject has received little attention in experimental hypothermia and clinical case reports. In order to explore the integrity of hypothermic and posthypothermic renal morphology we used an experimental animal model of accidental hypothermia where the heart supports the circulation throughout cooling and rewarming without accompanying cardioplegia or ischemia. Ultrastructural changes in renal tubular cells from three groups of pentobarbital anesthetized Wistar rats: 1) controls (n=6) maintained at 37 degrees C for 4 h, 2) hypothermic rats (n=6) core-cooled and maintained at 15-13 degrees C for 4 h, and 3) rewarmed rats (n=10), were studied as a sensitive indicator of renal damage. Electron micrographs (EM) from hypothermic kidneys showed rounded up mitochondria with loss of contrast. These changes were observed in several though not all of the biopsies, but they were found in all kidneys. Areas exhibiting focal tubular necrosis were seen on most EM from three of these kidneys. EM from rewarmed kidneys showed alterations of mitochondrial ultrastructure with similarities to those observed after hypothermia, but in general the changes were more prominent. Extracellular edema, intracellular edema, swelling of mitochondria, margination of chromatin, necrosis of single tubular cells, and disrupting necrotic debris into tubular lumen could be found in micrographs from 7 of the 10 kidneys examined. Rewarming from experimental hypothermia, without episodes of ischemia or hypoxia, thus induces ultrastructural changes in renal tubular cells similar to changes observed in acute tubular necrosis, associated with renal failure.

The effect of age and gender on epidermal nerve fiber density.

OBJECTIVE: Sensory neuropathies often involve small-diameter myelinated and unmyelinated nerve fibers, and neurologic and electrophysiologic findings may be normal unless larger nerve fibers are involved. The small (intra)epidermal nerve fibers (ENFs) now can be visualized with immunohistochemical techniques using the panaxonal marker anti-protein gene product 9.5 (PGP 9.5). Using this technique, the authors have established a reference range for ENF in a healthy white population and evaluated the reliability of the method. METHODS: Two punch biopsies, 3 mm in diameter, were taken from the distal part of the leg in 106 healthy volunteers (mean age, 49.0 +/- 19.6 years). Fifty-micrometer frozen thick sections were incubated with rabbit polyclonal antibodies to human PGP 9.5. The number of ENF/mm then was reported as the mean of counts in six sections (three sections from each of the two biopsies). RESULTS: The mean number of ENFs was 12.4 +/- 4.6 mm. In a multiple regression model, the density of ENF depended on age and gender (Y = 13.92 + 2.25 (gender) - 0.06 x age). The mean difference in ENF by intraobserver analysis was 0.2 +/- 1.2 ENF/mm, and by interobserver analysis, it was 0.4 +/- 1.5 fibers/mm. CONCLUSION: Normal means and ranges for the density of epidermal nerve fibers in a reference population have been established. The density of epidermal nerve fibers decreases with age and is lower in men compared with women. Intraobserver and interobserver analysis proves the reliability of the method.

Evolution of the nuclear modification factors with rapidity and centrality in d + Au collisions at (sqrt)[N(S)N]=200 GeV.

We report on a study of the transverse momentum dependence of nuclear modification factors R(dAu) for charged hadrons produced in deuteron + gold collisions at sqrt[s(NN)]=200 GeV, as a function of collision centrality and of the pseudorapidity (eta=0, 1, 2.2, 3.2) of the produced hadrons. We find a significant and systematic decrease of R(dAu) with increasing rapidity. The midrapidity enhancement and the forward rapidity suppression are more pronounced in central collisions relative to peripheral collisions. These results are relevant to the study of the possible onset of gluon saturation at energies reached at BNL RHIC.

Transverse-momentum spectra in Au+Au and d+Au collisions at sqrt[s(NN)]=200 GeV and the pseudorapidity dependence of high-p(T) suppression.

We present spectra of charged hadrons from Au+Au and d+Au collisions at sqrt[s(NN)]=200 GeV measured with the BRAHMS experiment at RHIC. The spectra for different collision centralities are compared to spectra from p+(-)p collisions at the same energy scaled by the number of binary collisions. The resulting ratios (nuclear modification factors) for central Au+Au collisions at eta=0 and eta=2.2 evidence a strong suppression in the high p(T) region (>2 GeV/c). In contrast, the d+Au nuclear modification factor (at eta=0) exhibits an enhancement of the high p(T) yields. These measurements indicate a high energy loss of the high p(T) particles in the medium created in the central Au+Au collisions. The lack of suppression in d+Au collisions makes it unlikely that initial state effects can explain the suppression in the central Au+Au collisions.

Does imprint cytology of brain tumours improve intraoperative diagnoses?

OBJECTIVES: To evaluate the diagnostic accuracy using frozen sections only and a combination of imprint cytology and frozen sections. MATERIAL AND METHODS: After introduction of imprint cytology as a supplement to frozen sections in 1999, 153 patients with brain tumours underwent stereotactic or open surgery. An equal number of cases prior to 1999 were chosen for comparison. Intraoperative diagnoses were compared with final diagnoses based on paraffin sections of the same tissue samples. The number of delayed intraoperative diagnoses was noted in each patient group. RESULTS: The combined use of the two techniques improved intraoperative diagnostic accuracy from 87 to 91% while the delayed intraoperative diagnoses were significantly reduced from 30 to 8. The choice of surgical procedure did not affect the outcome of the pathological investigations. CONCLUSION: A combination of frozen sections and imprints significantly reduced the number of delayed intraoperative diagnoses. Intraoperative diagnostic accuracy was improved, although not to a statistically significant level. Choice of surgical procedure did not affect the diagnostic outcome.

[Skin biopsy for quantification of intraepidermal nerve fibers]. / Hudbiopsi for kvantifisering av intraepidermale nervefibrer.

BACKGROUND: Skin biopsy for quantification of intraepidermal nerve fibre density has recently been introduced as a method for diagnosis of peripheral neuropathies. Immunostaining by antibody to protein gene product 9.5 has proved particularly useful because it selectively visualizes the epidermal nerve fibres. MATERIAL AND METHODS: We describe the procedure on the basis of relevant literature and our own experience. Results from investigations of 56 healthy individuals and three patients with small fibre involvement as part of their neuropathy are presented. RESULTS: In the healthy individuals, the mean density of epidermal fibres was 12.4 (SD 4.6), median 11.3 and range 6.0-26.1. Three patients with small fibre neuropathy had low intraepidermal nerve fibre density. INTERPRETATION: Skin biopsy for determination of intraepidermal nerve fibre density is a simple and non-painful procedure. Skin biopsies can be done repeatedly and may be used for the purpose of monitoring potential therapeutic agents.

Temporal bone histopathologic and genetic studies in Mohr-Tranebjaerg syndrome (DFN-1).

OBJECTIVE: To describe the temporal bone histopathologic and genetic abnormalities in a case of Mohr-Tranebjaerg syndrome. BACKGROUND: Mohr-Tranebjaezrg syndrome (DFN-1) is an X-linked, recessive, syndromic hearing loss, characterized by postlingual sensorineural hearing loss with onset in childhood, followed in adult life by progressive dystonia, spasticity, dysphagia, and optic atrophy. The syndrome is caused by mutations in the DDP (deafness/dystonia peptide) gene, which are thought to result in mitochondrial dysfunction with subsequent neurodegeneration. The temporal bone pathologic changes in this syndrome have not been reported. METHODS: Hearing loss developed in the patient at age 4, blindness at age 48, and dystonia at age 57. Genetic studies on peripheral blood showed a l51delT mutation in his DDP gene. He died at age 66. The right temporal bone was subjected to light microscopy and polymerase chain reaction-based analysis of the DDP gene sequence. RESULTS: There was near complete loss of spiral ganglion cells with loss of nearly all peripheral and central processes. Only 1,765 spiral ganglion cells remained (8.5% of mean normal for age). The organ of Corti (including hair cells), stria vascularis, and spiral ligament were preserved. There was also a severe loss of Scarpa's ganglion cells with preservation of vestibular hair cells. The population of geniculate and trigeminal ganglion cells appeared normal. Sequence analysis from temporal bone DNA showed the 15ldelT DDP gene mutation. CONCLUSION: Sensorineural hearing loss in Mohr-Tranebjaerg syndrome is the result of a postnatal, progressive, severe auditory neuropathy.

[Diagnostic transmission electron microscopy]. / Diagnostisk transmisjonselektronmikroskopi.

BACKGROUND: Transmission electron microscopy (TEM) is an important diagnostic tool in surgical pathology. MATERIAL AND METHODS: We give an overview, based on our own experience, of the application of TEM in diagnostic pathology. RESULTS: In spite of its potential for high-grade resolution, the actual use of TEM in diagnostic pathology is rather limited. TEM is mandatory in the examination of kidney and muscle biopsies and is often indicated when a metabolic disease is suspected. However, in many centres its importance in tumour pathology has declined because of the advances in the field of immunohistochemistry. TEM requires a great deal of resources and high levels of skills in tissue processing and diagnostic interpretation. INTERPRETATION: The TEM diagnosis must be integrated with light microscopical and, often, immunohistopathological findings, as well as with the clinical data. We therefore recommend close collaboration between the clinician and the laboratory with regard to biopsy indication, handling of samples, and the final diagnosis.

Neuronal cell death in the visual cortex is a prominent feature of the X-linked recessive mitochondrial deafness-dystonia syndrome caused by mutations in the TIMM8a gene.

The Mohr-Tranebjaerg syndrome (MIM 304700) and the Jensen syndrome (MIM 311150) were previously reported as separate X-linked recessive deafness syndromes associated with progressive visual deterioration, dystonia, dementia, and psychiatric abnormalities. In the most extensively studied Norwegian family, the Mohr-Tranebjaerg syndrome was reported to be caused by a one-basepair deletion (151delT) in the deafness/dystonia peptide (DDP) gene at Xq22. This gene has been renamed TIMM8a. We identified a stop mutation (E24X) in the TIMM8a gene segregating with the disease in the original Danish family with the Jensen syndrome, which confirms that the two disorders are allelic conditions. We also report abnormal VEP examinations and neuropathological abnormalities in affected males from the two unrelated families with different mutations. The findings included neuronal cell loss in the optic nerve, retina, striate cortex, basal ganglia, and dorsal roots of the spinal cord. The demonstration of mitochondrial abnormalities in skeletal muscle biopsies in some patients is compatible with the suggestion from recent research that the TIMM8a protein is the human counterpart of an intermembrane mitochondrial transport protein, Tim8p, recently characterized in yeast. The clinical and neuropathological abnormalities associated with mutations in the TIMM8a gene support that this X-linked deafness-dystonia-optic neuropathy syndrome is an example of progressive neurodegeneration due to mutations in a nuclear gene necessary for some, yet unknown mitochondrial transport function. We recommend sequencing the TIMM8a gene, thorough ophthalmological examination, and measuring visual evoked potentials in clinically suspected male patients with either progressive hearing impairment, dystonia, or visual disability in order to establish an early diagnosis and provide appropriate genetic counselling.

A new congenital myopathy in a Norwegian family.

UNLABELLED: A 6-y-old boy presented with a mild, and apparently non-progressive, congenital myopathy, primarily affecting explosive movements such as running and jumping. Five other cases, spanning four generations, were identified in his family. A dominant inheritance pattern was suggested. Quadriceps muscle histology showed a selective type II fibre atrophy, which is otherwise considered a non-specific change associated with a number of conditions. CONCLUSION: A Norwegian boy with an inherited muscle weakness is presented. Based on clinical and laboratory investigations, and in light of the inheritance pattern, a previously undescribed congenital myopathy is suggested.

Leucovorin and maximum tolerated dose toxicity of methotrexate in rats.

High-dose methotrexate (HD-MTX) is widely used in combination chemotherapy and can be handled without life-threatening toxicity in combination with leucovorin (LV) rescue. However, in an experimental animal model for testing of short-term HD-MTX effects in anesthetized rats, the authors previously demonstrated intolerable toxicity and death within a few hours in some animals. Serum levels were below levels routinely found in patients on HD-MTX treatment. This study was aimed at disclosure of possible mechanisms for acute toxicity of MTX in rats. The previously determined maximum tolerated dose of 5 g/kg MTX was used as the test dose. The animals that died showed sudden reduction in heart rate and blood pressure. LV, 1 g/kg infused immediately prior to MTX, changed MTX elimination kinetics, but did not change the acute toxicity. The data of this study together with additional evidence obtained in the experimental model, suggest that MTX acute toxicity may not be related to its antiproliferative effect, but rather to perturbation of endothelial cell and platelet function.

Microvascular perturbations in rats receiving the maximum tolerated dose of methotrexate or its major metabolite 7-hydroxymethotrexate.

Methotrexate (MTX) is a clinically important cytostatic antifolate. The study describes the acute effects of maximum tolerated doses of MTX or its major metabolite 7-hydroxymethotrexate (7-OH-MTX) on the ultrastructure of rat liver and kidneys. The ultrastructural changes in rats receiving MTX or 7-OH-MTX were, in principle, indistinguishable and their severity and extension increased with time of survival or doses of medication. All lesions were focal, microvascular, or parenchymal. Microvascular changes were more severe in nature when blood cells were present. The endothelial cells were swollen with loss of pinocytotic vesicles, their luminal plasma membrane formed blebs or were disrupted. Partly detached endothelial cells or deendothelialized areas, various types of white blood cells, in particular, neutrophil granulocytes, were observed in the microcirculation. Single platelets or small platelet aggregates were found either in the lumen or adhering to deendothelialized areas of injured endothelial cells. Hepatocytes exhibited steatosis, edema, and manifest single cell necrosis. There were also nuclear changes, marked proliferation of smooth endoplasmatic reticulum, increased amounts of intracellular lipid vacuoles, and a decrease in glycogen particles in hepatocytes. The kidney presented the major changes in the tubules and in the interstitial part. MTX and 7-OH-MTX acute toxicity may primarily be related to microvascular perturbation.