Mila Ćetković

Ultrastructure and immunohistochemistry of the trigeminal peripheral myelinated axons in patients with neuralgia

OBJECTIVE Detailed ultrastructural and immunohistochemical examination of the trigeminal axons surrounded by the peripheral type of the myelin could add new information about the extent of the trigeminal nerve lesion in neuralgia. PATIENTS, MATERIALS AND METHODS The examination comprised, firstly, the 10 trigeminal nerve roots (TNRs) in which the neurovascular contact was found in 20% of the cases, and the 2 additional control TNRs. Secondly, the biopsy specimens were taken from 6 patients with trigeminal neuralgia and 2 patients with trigeminal neuropathy following a partial TNR rhizotomy. The specimens were examined under the electron microscope (EM) and/or using the immunohistochemical (IHC) methods. RESULTS In addition to the central zone of demyelination, the EM examination of the TNR also revealed alterations of the peripheral myelin, i.e. deformation, thickening, demyelination and remyelination, as well as changes of the peripheral axons, that is, atrophy or hypertrophy, neurofilaments increase, loss of the myelin and sprouting occasionally. Some Schwann cells were also damaged. The IHC examination usually showed a moderate immune reaction against neuron-specific enolase (NSE) and protein gene product 9.5 (PGP9.5), but sporadically weaker reaction against the S-100 protein, synaptophysin (SY), neurofilament protein (NFP) and glial fibrillary acidic protein (GFAP). The substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactivity was weak at some sites, but strong at some other places. CONCLUSIONS The pathological changes affect not only the central nerve fibers of the TNR, but also some of the peripheral axons, their myelin sheath and Schwann cells. These are signs of the retrograde ultrastructural and biochemical alterations, which could participate in the pathophysiological mechanism underlying the trigeminal neuralgia.

The Trigeminal Vasculature Pathology in Patients With Neuralgia

Objective.—To examine the possible pathological changes of the trigeminal vasculature in patients with neuralgia.

Fractal analysis and Gray level co-occurrence matrix method for evaluation of reperfusion injury in kidney medulla.

Fractal analysis and Gray level co-occurrence matrix method represent two novel mathematical algorithms commonly used in medical sciences as potential parts of computer-aided diagnostic systems. In this study, we tested the ability of these methods to discriminate the kidney medullar tissue suffering from reperfusion injury, from normal tissue. A total of 320 digital micrographs of Periodic acid-Schiff (PAS) - stained kidney medulla from 16 Wistar albino mice (20 per animal), were analyzed using National Institutes of Health ImageJ software (NIH, Bethesda, MD) and its plugins. 160 micrographs were obtained from the experimental group with induced reperfusion injury, and another 160 were obtained from the controls. For each micrograph we calculated the values of fractal dimension, lacunarity, as well as five GLCM features: angular second moment, entropy, inverse difference moment, GLCM contrast, and GLCM correlation. Discriminatory value of the parameters was tested using receiver operating characteristic (ROC) analysis, by measuring the area below ROC curve. The results indicate that certain features of GLCM algorithm have excellent discriminatory ability in evaluation of damaged kidney tissue. Fractal dimension and lacunarity as parameters of fractal analysis also had a relatively good discriminatory value in differentiation of injured from the normal tissue. Both methods have potentially promising application in future design of novel techniques applicable in cell physiology, histology and pathology.

Morphometric analysis, region of supply and microanatomy of the lenticulostriate arteries and their clinical significance.

The lenticulostriate arteries (LSA) and their microanatomy, region of supply and atherosclerosis were examined in 24 microdissected brains, arterial casts, and histological specimens. The LSA ranged from 2 to 12 in number and from 0.10 mm to 1.28 mm in diameter. They always arose from the initial segment of the middle cerebral artery (MCA), often from the MCA leptomeningeal branches (38.24%), and rarely from the insular segment (2.94%). They always originated as individual branches, often (61.76%) with their own common stems. In two hemispheres we found that the LSA supplied either a larger or a smaller portion of the basal ganglia and internal capsule than usual. The number of twigs to the innominate substance (substantia innominata) (3-11), and their diameters (0.07-0.30 mm), has been described for the first time, to our knowledge. Microatheromas were found in two LSA. Data about the LSA microanatomy and territory could form the basis of safer neurosurgery, more accurate neuroimaging evaluation, and precise neurological diagnosis in patients with focal ischemic lesions in the basal ganglia and internal capsule.

The acidophilic nature of neuronal Golgi impregnation.

The mechanisms of Golgi impregnation of neurons has remained enigmatic for decades. Recently, it was suggested that divalent (di)chromate anions play a role in the Golgi impregnation process. Therefore, we incubated slices of (para)formaldehyde-fixed rat brain tissue in solutions of potassium (di)chromate, phosphate, chloride or nitrate at pH 6 or 7. Slices were then immersed in solutions of silver nitrate and processed for light microscopical analysis. At pH 6, dichromate probes resulted in dense and homogeneous impregnation of neuronal cytoplasm (typical impregnation). At pH 7, chromate probes showed solely partial cytoplasmic and heavy nuclear-region neuron impregnation (atypical impregnation). Phosphate probes at pH 6 resulted in typical impregnation, whereas at pH 7 phosphate probes gave atypical impregnation. Both at pH 6 and 7, chloride and nitrate probes did not yield any Golgi impregnation. These findings confirmed the pH-dependence of silver-chromate Golgi impregnation as well as the correctness of corresponding acidic silver-phosphate impregnation. Our study revealed a previously unknown, strong anion-dependence of Golgi impregnation, suggesting that hydrogenated monovalent anions are carriers of the neuron impregnation.

Chromatin Fractal Organization, Textural Patterns, and Circularity of Nuclear Envelope in Adrenal Zona Fasciculata Cells

Despite previous research efforts in the fields of histology and cell physiology, the relationship between chromatin structural organization and nuclear shape remains unclear. The aim of this research was to test the existence and strength of correlations between mathematical parameters of chromatin microarchitecture and roundness of the nuclear envelope. On a sample of 240 nuclei of adrenal zona fasciculata cells stained using the DNA-specific Feulgen method, we quantified fractal parameters such as fractal dimension and lacunarity, as well as textural parameters such as angular second moment (ASM), entropy, inverse difference moment, contrast, and variance. Circularity of the nuclear envelope was determined from the nuclear area and perimeter. The results indicate that there is a statistically significant negative correlation between chromatin ASM and circularity. Moreover, there was a statistically significant positive correlation between chromatin fractal dimension and envelope circularity. This is the first study to demonstrate these relationships in adrenal tissue, and also one of the first studies to test the connection between circularity and fractal and gray-level co-occurrence matrix parameters in DNA-specific Feulgen stain. The results could be useful both as an addition to the current knowledge on chromatin/nuclear envelope interactions, and for design of future computer-assisted research software for evaluation of nuclear morphology.

Immunohistochemistry of Displaced Sensory Neurons in the Trigeminal Nerve Root

The aim of this study was to examine the morphology and the immunohistochemical features of displaced ganglion cells in the trigeminal nerve root (TNR). Forty human TNRs of 20 persons, obtained during routine autopsy in accordance with the Declaration of Helsinki, were examined following Klüver-Barrera and azan trichrome histological staining, and immunohistochemical reactions against certain neuronal markers, neuropeptides and neurotransmitters. A total number of 61 displaced neurons were investigated, which were present in 80% of individuals studied. Displaced neurons were found in 55.0% of the TNRs, either in the sensory portion (22.5%), motor portion (22.5%) or both (10.0%). Neuronal diameter varied from 12.5 × 25.0 to 45.0 × 63.7 (mean 27.6 × 41.6) µm, and in area between 245 and 2,065 (mean 927) µm2. Each neuron was surrounded by 2–17 elongated satellite cells per slice. The immune reaction was positive in all the neurons studied for neuron-specific enolase, protein gene product 9.5, neurofilament protein and synaptophysin, and in some neurons for calcitonin gene-related peptide (CGRP; 24.4%), cholecystokinin (CCK; 13.3%), somatostatin (SST; 17.8%), substance P (SP; 15.6%), vasoactive intestinal polypeptide (4.4%), neuropeptide Y (8.9%), and serotonin (11.1%). The immune reactions were most frequent against the CGRP, SP, CCK and SST. We concluded that displaced neurons in the TNR morphologically and immunohistochemically resembled the sensory neurons in the trigeminal ganglion.

Blood supply of the trigeminal ganglion and nerve root

Abstract Great surgical significance and lack of relevant anatomic data were the reasons for this study. Twenty-five trigeminal nerve roots and ganglia were examined under the stereoscopic microscope. The nerve root received between two and six vascular twigs from two or three of the following arteries: the superolateral pontine (92%), anterior inferior cerebellar (88%), inferolateral or posterolateral pontine, superior cerebellar, basilar and trigeminocerebellar. The trigeminal twigs measured from 110 to 520 μm in diameter. A single trigeminal artery may supply either the motor portion of the nerve root or the sensory portion, or both. The superolateral pontine artery (88%) usually perfused the motor root. The same artery often supplied (64%) the ophthalmic part of the sensory root. The maxillary part was most often irrigated by the superolateral and inferolateral pontine arteries, and the mandibular part by the anterior inferior cerebellar artery (AICA). The trigeminal ganglion received the vascular twigs from the middle meningeal artery (92%), accessory middle meningeal artery (8%), inferolateral trunk (90%) and tentorial branch (8%). The obtained data in the trigeminal vasculature can be the anatomic basis for decompressive neurovascular operations and surgery of the cavernous sinus.

Toxicity evaluation of two polyoxotungstates with anti-acetylcholinesterase activity

ABSTRACT A toxicity evaluation of two Keggin‐type heteropolytungstates, K7[Ti2PW10O40]·6H2O and K6H[SiV3W9O40]·3H2O, with different inhibitory potencies toward acetylcholinesterase activity (IC50 values of 1.04 × 10− 6 and 4.80 × 10− 4 mol/L, respectively) was performed. Wistar albino rats were orally treated with single doses (5 and 50 mg/kg) of both investigated compounds. The biochemical parameters of renal (serum urea and creatinine) and liver function (direct and total bilirubin, alanine transaminase, and aspartate aminotransferase) were determined after 24 h and 14 days. A histopathological analysis of liver tissue was carried out 14 days after the polyoxotungstate administration. Both applied doses of the investigated compounds did not induce statistically significant alterations of the renal function markers. However, the polyoxotungstate treatment caused an increase in the activities of serum alanine transaminase and aspartate aminotransferase in a time‐ and concentration‐dependent manner, although statistically significant changes in bilirubin concentrations were not observed. Furthermore, the detected hepatotoxic effect was confirmed by histhopathological analysis that suggested some reversible liver tissue damage two weeks after the treatment, especially in the case of K6H[SiV3W9O40]·3H2O. Accordingly, the toxicity of these two polyoxotungstates with anti‐acetylcholinesterase effect cannot be considered as a severe one, but their potential clinical application would require a more complex toxicological study. HighlightsK‐Ti2PW10 and K‐SiV3W9in vitro inhibit AChE activity with different potencies.Both K‐Ti2PW10 and K‐SiV3W9 do not influence rat body mass and food intake.Both K‐Ti2PW10 and K‐SiV3W9 do not significantly alter biomarkers of renal function.K‐Ti2PW10 and K‐SiV3W9 significantly increase hepatotoxicity parameters, ALT and AST.K‐Ti2PW10 and K‐SiV3W9 cause reversible liver tissue damage 2 weeks after application.

White Matter Changes in Two Leber's Hereditary Optic Neuropathy Pedigrees: 12-Year Follow-Up.

We are presenting two Lebers hereditary optic neuropathy (LHON) pedigrees with abnormal magnetic resonance imaging (MRI) and proton magnetic resonance spectroscopy (H-MRS) findings but without neurological manifestation associated with LHON. The study included 14 LHON patients and 41 asymptomatic family members from 12 genealogically unrelated families. MRI showed white matter involvement and H-MRS exhibited metabolic anomalies within 12 LHON families. Main outcome measures were abnormal MRI and H-MRS findings in two pedigrees. MRI of the proband of the first pedigree showed a single demyelinating lesion in the right cerebellar hemisphere, while the proband of the second family displayed multiple supratentorial and infratentorial lesions, compatible with the demyelinating process, and both the absolute choline (Cho) concentration and Cho/creatinine ratio were increased. MRI and H-MRS profiles of both affected and unaffected mitochondrial DNA mutation carriers suggest more widespread central nervous involvement in LHON. Although even after 12 years our patients did not develop neurological symptoms, MRI could still be used to detect possible changes during the disease progression.