Luxol Fast Blue-cresyl Violet Research Articles

Histochemical staining techniques in Culex pipiens and Drosophila melanogaster (Diptera) with a comparison to mammals.

Insects play an important role in ecosystems. Changes in their abundance and biodiversity are of paramount interest, as there has not only been an alarming decline of insects important for ecosystem health throughout the past decades, but also an increase in insects detrimental for biomes. Furthermore, insects pose a threat to modern society as arbovirus-transmitting vectors. Therefore, detailed knowledge of insect staining characteristics could be beneficial as a basis for further studies, whether in the context of species conservation or control of insect pests. Thus, this study compared 14 histochemical stains for their usefulness in insects regarding nervous tissue, connective tissue components, mucins and polysaccharides, mineralization, and microorganisms. The study used formalin-fixed paraffin-embedded tissue sections of mammals (Equus caballus) and 2 dipterans (Culex pipiens biotype molestus, Drosophila melanogaster). Several histochemical stains were suitable for tissue assessment in insects and mammals, in particular for nervous tissue (Bielschowsky silver stain, luxol fast blue-cresyl violet) and polysaccharides (alcian blue, periodic acid-Schiff with and without diastase treatment, toluidine blue). Other stains proved useful for visualization of insect-specific organ characteristics such as Gomori's reticulin stain for tracheoles in both dipteran species, Heidenhain's azan for midgut-associated connective tissue, and von Kossa for mineral deposition in Malpighian tubules of C. pipiens biotype molestus. In summary, this study provides comparable insights into histochemical procedures in mammals and insects and their usefulness for histological assessment of C. pipiens biotype molestus and D. melanogaster.

Impact of Astrocyte Depletion upon Inflammation and Demyelination in a Murine Animal Model of Multiple Sclerosis.

Astrocytes play a key role in demyelinating diseases, like multiple sclerosis (MS), although many of their functions remain unknown. The aim of this study was to investigate the impact of astrocyte depletion upon de- and remyelination, inflammation, axonal damage, and virus distribution in Theiler’s murine encephalomyelitis (TME). Groups of two to six glial fibrillary acidic protein (GFAP)-thymidine-kinase transgenic SJL mice and SJL wildtype mice were infected with TME virus (TMEV) or mock (vehicle only). Astrocyte depletion was induced by the intraperitoneal administration of ganciclovir during the early and late phase of TME. The animals were clinically investigated while using a scoring system and a rotarod performance test. Necropsies were performed at 46 and 77 days post infection. Cervical and thoracic spinal cord segments were investigated using hematoxylin and eosin (H&E), luxol fast blue-cresyl violet (LFB), immunohistochemistry targeting Amigo2, aquaporin 4, CD3, CD34, GFAP, ionized calcium-binding adapter molecule 1 (Iba1), myelin basic protein (MBP), non-phosphorylated neurofilaments (np-NF), periaxin, S100A10, TMEV, and immunoelectron microscopy. The astrocyte depleted mice showed a deterioration of clinical signs, a downregulation and disorganization of aquaporin 4 in perivascular astrocytes accompanied by vascular leakage. Furthermore, astrocyte depleted mice showed reduced inflammation and lower numbers of TMEV positive cells in the spinal cord. The present study indicates that astrocyte depletion in virus triggered CNS diseases contributes to a deterioration of clinical signs that are mediated by a dysfunction of perivascular astrocytes.

Histopathological characterization of Toxocara canis- and T. cati-induced neurotoxocarosis in the mouse model.

Infective larvae of Toxocara canis and T. cati, the common roundworms of dogs and cats, may invade the central nervous system of paratenic hosts, including humans, causing neurotoxocarosis (NT). Previous studies on NT in the model organism "mouse" have indicated distinct differences between T. canis and T. cati regarding larval migration patterns as well as the severity of clinical symptoms and behavioural alterations. The objective of the present study was to provide an extensive characterization of the underlying histopathological alterations, comparing T. canis- and T. cati-induced changes in different brain areas over the course of murine infection. Four histological sections of five brains each of T. canis- and T. cati-infected as well as uninfected C57Bl/6 mice were investigated 7, 14, 28, 42, 70 and 98days post infection (dpi), while brains of T. cati-infected and control mice were also available 120 and 150 dpi. In addition to haematoxylin-eosin and luxol fast blue-cresyl violet staining, immunohistochemistry was employed to study microglia/macrophage cell morphology and to detect accumulation of β-amyloid precursor protein (β-APP) as an indicator of axonal damage. Haemorrhages, eosinophilic vasculitis and activated microglia/macrophages were detected in both infection groups starting 7 dpi, followed by eosinophilic meningitis in cerebra as from 14 dpi. Overall, little differences in the proportion of animals affected by these alterations were found between the two infection groups. In contrast, the proportion of animals displaying β-APP accumulation was significantly higher in the T. canis than T. cati group as from 28 dpi regarding the cerebrum as well as at 98 dpi regarding the cerebellum. In T. canis-infected mice, myelinophagic microglia/macrophages ("gitter cells") appeared as from 14 dpi, whereas these were first observed at 70 dpi in T. cati-infected animals. The proportion of animals displaying demyelination and/or gitter cells in the cerebrum was significantly higher in the T. canis than T. cati group as from 28 dpi, and at 28 and 42 dpi regarding the cerebellum. Earlier and more severe neurodegeneration during T. canis- than T. cati-induced NT, especially in the cerebrum, may explain the differences in behavioural alterations observed in previous studies. In addition to differences in larval migration preferences, immunological processes may contribute to these patterns, which warrant further investigation.

Effect of Multiple Intraperitoneal Injections of Human Bone Marrow Mesenchymal Stem Cells on Cuprizone Model of Multiple Sclerosis

Background:Bone marrow mesenchymal stem cells (BM-MSCs) elicit neuroprotective effects, and their repair ability has been investigated in different experimental models. We aimed to investigate the effect of multiple i.p. BM-MSCs injections in the cuprizone model of multiple sclerosis in mice.Methods:Adult male C57BL/6 mice (n = 40) were fed a regular diet or a diet containing cuprizone (0.2% w/w) for six weeks. Bone marrow samples were taken from patients with spinal cord injury. BM-MSCs (2 × 106 in 1 milliliter medium) were administered intraperitoneally for two consecutive weeks at the end of the forth weeks of cuprizone administration. Animals (n = 12) were perfused with 10% paraformaldehyde at the end of sixth week. The brains were sectioned coronally in 6-8-μm thickness (-2.3 to 1.8 mm from bregma). The sections were stained by luxol fast blue-cresyl violet, and images were captured via a microscope. Demyelination ratio was estimated in corpus callosum in a blind manner. A quantitative real-time PCR was used to measure the myelin basic protein gene expression at sixth week.Results:Histologically, cuprizone induced demyelination in the corpus callosum. Demyelinated area was diminished in the corpus callosum of cell-administered group. Cuprizone could decrease myelin-binding protein mRNAs expression in corpus callosum, which was significantly recovered after BM-MSCs injections.Conclusion:Our data indicated a remyelination potency of multiple i.p. BM-MSCs in the cuprizone model of multiple sclerosis in mice.

Viruses associated with congenital tremor and high lethality in piglets.

The recently described atypical porcine pestivirus (APPV) has been associated with congenital tremor (CT) type A-II in piglets in different countries. Another important neurological pathogen of pigs is porcine teschovirus (PTV), which has been associated with non-suppurative encephalomyelitis in pigs with severe or mild neurological disorders. There have been no reports of APPV and/or PTV coinfection associated with CT or encephalomyelitis in Brazilian pig herds. The aim of this study was to describe the pathological and molecular findings associated with simultaneous infection of APPV and PTV in piglets with clinical manifestations of CT that were derived from a herd with high rates of CT-associated lethality. In 2017, three piglets from the same litter with CT died spontaneously. The principal pathological alterations in all piglets were secondary demyelination and hypomyelination at the cerebellum, brainstem and spinal cord confirmed by histopathology and luxol fast blue-cresyl violet stain. Additional significant pathological findings included multifocal neuronal necrosis, neuronophagia and gliosis found in the cerebral cortex and spinal cord of all piglets, while atrophic enteritis and mesocolonic oedema were observed in some of them. APPV and PTV RNA were detected in the central nervous system of affected piglets, and PTV was also detected in the intestine and faeces. The pathological alterations and molecular findings together suggest a dual infection due to APPV and PTV at this farm. Moreover, the combined effects of these pathogens can be attributed to the elevated piglet mortality, as coinfections involving PTV have a synergistic effect on the affected animals.

High resolution MR anatomy of the subthalamic nucleus: Imaging at 9.4T with histological validation

Using conventional MRI the subthalamic nucleus (STN) is not clearly defined. Our objective was to define the anatomy of the STN using 9.4 T MRI of post mortem tissue with histological validation. Spin-echo (SE) and 3D gradient-echo (GE) images were obtained at 9.4 T in 8 post mortem tissue blocks and compared directly with corresponding histological slides prepared with Luxol Fast Blue/Cresyl Violet (LFB/CV) in 4 cases and Perl stain in 3. The variability of the STN anatomy was studied using internal reference points. The anatomy of the STN and surrounding structures was demonstrated in all three anatomical planes using 9.4 T MR images in concordance with LFB/CV stained histological sections. Signal hypointensity was seen in 6/8 cases in the anterior and medial STN that corresponded with regions of more intense Perl staining. There was significant variability in the volume, shape and location of the borders of the STN. Using 9.4 T MRI, the internal signal characteristics and borders of the STN are clearly defined and significant anatomical variability is apparent. Direct visualisation of the STN is possible using high field MRI and this is particularly relevant, given its anatomical variability, for planning deep brain stimulation.

Temporal profile of T2-Weighted MRI Distinguishes between Pannecrosis and Selective Neuronal Death after Transient Focal Cerebral Ischemia in the Rat

Transient middle cerebral artery occlusion (MCAO) by an intraluminal thread leads to primarily subcortical infarctions with little sensorimotor impairment in the Wistar rat strain. We investigated the course of infarct development in this lesion type for 10 weeks using magnetic resonance imaging (MRI) along with histological characterization. MCAO was induced in male Wistar rats (260 to 300 g) for 60 mins. Animals received follow-up T1- and T2-weighted MRI from day 1 until week 10. Separate groups of animals were analyzed histologically after 2, 6, and 10 weeks. Histology included immunohistochemistry for neuronal and astrocytic markers as well as hematoxylin eosin and luxol fast blue-cresyl violet staining. In contrast to lesions involving the cortex, exclusively subcortical infarctions were characterized by a complete resolution of initially increased T1 and T2 relaxation times by 10 weeks. Between 2 and 10 weeks, neuronal death and gliosis as well as a dense inflammatory infiltrate were evident in these lesions, without damage to fiber tracts or development of cystic cavities. Exclusively subcortical lesions in Wistar rats are characterized by normalization of T1 and T2 relaxation times, which might, however, not be mistaken for tissue recovery. Despite this MRI normalization, selective neuronal death and gliosis develop. Although MRI at individual time points might therefore be ambiguous, the temporal profile of relaxation time changes over the chronic time period allows discrimination of the lesion development into selective neuronal death or pannecrosis.

Cochlear nuclear complex of mice.

Using serial sections stained with luxol fast blue-cresyl violet, the cochlear nuclei of CBA/J mice were parcellated into the same cytoarchitectonic areas and layers that Osen (1969) used in cats. Within the spherical cell areas, the distribution of Nissl substance is more reliable than soma shape in identifying the spherical cells. The area of large spherical cells is extremely small in CBA/J mice but does contain significantly larger neurons than the small spherical cell area. In Golgi preparations, bushy cells are found in all areas of the ventral cochlear nucleus except in the octopus cell area and granule cell layer. They are more numerous anteriorly than posteriorly and details of their morphology are quite variable. Stellate cells are found throughout the ventral cochlear nuclei but are present in greatest numbers in the multipolar cell area; they are rare in the large spherical cell area and octopus cell area. Because size, soma morphology, and dendritic arborization vary on a continuum rather than in discrete steps, we have not subcategorized these neurons. Octopus cells are restricted to the posterior, dorsomedial area of the ventral cochlear nucleus. In the central region of the dorsal cochlear nucleus, stellate cells abound and are categorized as vertical, elongate, or radiate cells. In the granule layer of the dorsal cochlear nucleus there are both fusiform and Purkinje-like neurons, so named because of their resemblance to cerebellar Purkinje cells. These Purkinje-like cells differ from fusiform cells in having 1) a smaller cell body, spherical in shape, 2) no basal dendrites, 3) a sagittal dendritic orientation, 4) elaborate dendritic branching, and 5) abundant dendritic spines.