Dynamics of pathomorphological changes of ultrastructural organization of intervertebral disc at different terms of experimental opioid influence and with its cancellation

Background. Patients with degenerative diseases of the spine - "difficult patients". Often, exhausted by long-term pain, they have a dependence, and sometimes tolerance to various analgesics, which greatly reduces the effectiveness of routinely used in the hospital anesthesia. Objective. The aim of our work was to study the features of pathomorphological manifestations of the structural components of the intervertebral disc at different terms of opioid influence and at the difference at the ultrastructural level in the experiment. Methods. The material of the study were sexually mature, white, nonlinear rats - males in the amount of 90 hundred animals, weighing 92 - 103 g, aged 4.5 months. Animals were injected with Nalbuphine at home, once daily for one day (10-11 am) for 42 days. The initial dose of Nalbuphine was 8 mg / kg during the first week, 15 mg / kg during the second week; 20 mg / kg during the third week; 25 mg / kg during the fourth week; 30 mg / kg during the fifth week and 35 mg / kg during the sixth week of the experimental opioid effect. Thus created the conditions for chronic opioid exposure. Intervertebral discs of rats were used as material for ultrastructural study. Results and conclusion. As a result of our study, we found that at the end of 21 days we found the progression of alternative changes in the cellular elements of the gelatinous nucleus, characterized by the development of necrotic changes in notochondral cells, as well as chondroptosis of chondrocytes. Preserved notochondral cells were rarely visualized. In some places, there were notochondral cells in which the nucleus disintegrated into separate fragments filled with heterochromatin, and the remains of organelle membranes were localized in the enlightened cytoplasm. Pronounced destructive changes were found in chondrocytes. After 28 days, the changes progressed, this was manifested by the fact that in the pulpal nucleus there were extensive cell-free zones filled with a significant amount of granular intensely osmophilic mass. Notochondral cells and most chondrocytes underwent necrotic changes. After 35 days at the ultrastructural level revealed pronounced changes in the structural elements of the gelatinous nucleus and fibrous ring. Extensive cell-free zones were localized in the gelatinous nucleus, with a somewhat compacted matrix in which an intensely osmophilic fine-grained deep mass accumulated. With the abolition of opioid exposure at the end of 56 days, we found pronounced changes in notochondral cells and in the vast majority of chondrocytes. The matrix of the gelatinous nucleus was compacted, it showed thickened collagen fibrils. Most of the cellular elements of the gelatinous nucleus were at different stages of necrosis, and some chondrocytes - chondroptosis. Vacuoles filled with enlightened contents also appeared in the cytoplasm. The nucleus was compacted, condensation of chromatin was observed. In such areas, the fibrils of collagen fibers were loose, stratified, disintegrated and lysed.

EVIDENCE OF APOPTOSIS IN PARVOCELLULAR NUCLEI OF HYPOTHALAMUS IN DIABETES MELLITUS

Diabetes mellitus (DM) causes great socio-economic damage, which is determined by medical expenses and expenditure on social security to patients due to invalidity and loss of labour capacity. Researchers are studying the role of hypothalamic neuropeptides and their involvement in the regulation of pancreatic islet function. In view of the above, the aim of our study was to establish the features of morphofunctional changes in the arcuate nucleus (AN) and ventromedial nucleus (VN) of the hypothalamus in streptozotocin-induced diabetes mellitus (SIDM). Histological, immunohistochemical, electron-microscopic, biochemical and statistical research methods were used. Polymorphic changes were noted in AN and VN at early stages of development of SIDM (on the 14th day). In AN, the area of neurons and their nuclei became larger, the numerical density of dark functionally more active neurons increased, and dinuclear light neuroendocrine cells (NC) appeared. In light and dark NCs of AN, there was a significant increase in the bulk density of neurosecretory granules – by 2-4 times compared with the control, in VN – by 1.2-2 times. Such morphofunctional changes in parvocellular nuclei of the hypothalamus and the increase in the bulk density of neurosecretory granules in their NCs indicate boosted synthesis of neurohormones which directly affect the adenohypophysis and improving functional activity of NCs in AN and VN. On the 70th day of SIDM in AN and VN of the hypothalamus, there was a decrease in the numerical density of neurons due to light NC and increase in the numerical density of vacuolated, dark pycnomorphic and apoptotic neurons. The apoptotic index in the studied nuclei of the hypothalamus increased by 2-4 times compared with the control. The area of the profile field of NC increased and the area of nuclei decreased, which led to the reduced nuclear-cytoplasmic index and indicated a decrease in their functional activity and was confirmed by a decrease in the bulk density of neurosecretory granules in NC by 1.9-2.1 times in AN and by 2.7-4.7 times in VN. At the ultrastructural level, pronounced destructive processes such as vacuolar dystrophy, development of satelliteosis and neuronophagia were observed in light NCs. Thus, prolonged hyperglycemia in SIDM in parvocellular nuclei of the hypothalamus causes neuronal death to a lesser extent due to apoptosis, and to a greater extentdue due to hydropic dystrophy and colliquative necrosis, especially in the long term of the experiment (on the 70th day), and leads to the development of diabetic neuroendocrinopathy.

Ultrastructural Analysis of Neuroimplant-Parenchyma Interfaces Uncover Remarkable Neuroregeneration Along-With Barriers That Limit the Implant Electrophysiological Functions

Despite increasing use of in vivo multielectrode array (MEA) implants for basic research and medical applications, the critical structural interfaces formed between the implants and the brain parenchyma, remain elusive. Prevailing view assumes that formation of multicellular inflammatory encapsulating-scar around the implants [the foreign body response (FBR)] degrades the implant electrophysiological functions. Using gold mushroom shaped microelectrodes (gMμEs) based perforated polyimide MEA platforms (PPMPs) that in contrast to standard probes can be thin sectioned along with the interfacing parenchyma; we examined here for the first time the interfaces formed between brains parenchyma and implanted 3D vertical microelectrode platforms at the ultrastructural level. Our study demonstrates remarkable regenerative processes including neuritogenesis, axon myelination, synapse formation and capillaries regrowth in contact and around the implant. In parallel, we document that individual microglia adhere tightly and engulf the gMμEs. Modeling of the formed microglia-electrode junctions suggest that this configuration suffice to account for the low and deteriorating recording qualities of in vivo MEA implants. These observations help define the anticipated hurdles to adapting the advantageous 3D in vitro vertical-electrode technologies to in vivo settings, and suggest that improving the recording qualities and durability of planar or 3D in vivo electrode implants will require developing approaches to eliminate the insulating microglia junctions.

Ultrastructural analysis of neuroimplant-parenchyma interfaces uncover remarkable neuroregeneration along-with barriers that limit the implant electrophysiological functions

Despite increasing use of in-vivo multielectrode array (MEA) implants for basic research and medical applications, the critical structural interfaces formed between the implants and the brain parenchyma, remain elusive. Prevailing view assumes that formation of multicellular inflammatory encapsulating-scar around the implants (the foreign body response) degrades the implant electrophysiological functions. Using gold mushroom shaped microelectrodes (gMμEs) based perforated polyimide MEA platforms (PPMPs) that in contrast to standard probes can be thin sectioned along with the interfacing parenchyma; we examined here for the first time the interfaces formed between brains parenchyma and implanted 3D vertical microelectrode platforms at the ultrastructural level. Our study demonstrates remarkable regenerative processes including neuritogenesis, axon myelination, synapse formation and capillaries regrowth in contact and around the implant. In parallel, we document that individual microglia adhere tightly and engulf the gMμEs. Modeling of the formed microglia-electrode junctions suggest that this configuration suffice to account for the low and deteriorating recording qualities of in vivo MEA implants. These observations help define the anticipated hurdles to adapting the advantageous 3D in-vitro vertical-electrode technologies to in-vivo settings, and suggest that improving the recording qualities and durability of planar or 3D in-vivo electrode implants will require developing approaches to eliminate the insulating microglia junctions.

Dynamics of changes in the ultrastructural characteristics of the mucosa and cartilage of the larynx of rats at the end of 35 and 42 days of experimental opioid exposure and after 56 days with opioid withdrawal

In our work, we studied the ultrastructural organization of rat laryngeal mucosa and cartilage at the end of 35 and 42 days of experimental opioid exposure and at the end of 56 days with opioid withdrawal. As a result of our study at the end of the 35th day at the ultrastructural level revealed hypersecretion of goblet cells, mucus contained impurities of lymphocytes, single neutrophils and erythrocytes. In the enlarged lumens of the hemocapillaries of the lamina propria of the mucous membrane and submucosal base of different parts of the larynx, the adhesion of erythrocytes was noted. At the end of 42 days in rats, the changes were characterized by moderate damage to cytoplasmic organelles in epitheliocytes. Alternative changes developed in the chondrocytes of elastic and hyaline cartilages. Marked by an increase in the number of active fibroblasts, next to which were numerous fibrils of collagen fibers. The main substance of connective tissue, near dilated vessels, was impregnated with transudate, which was accompanied by the development of perivasal edema. Glycogen grains and vacuoles filled with neutral fats were visualized in the cytoplasm of chondrocytes.At the end of 56 days in rats with two-week withdrawal of the opioid analgesic, we found the development of alternative processes in the epitheliocytes of different parts of the larynx, as well as in chondrocytes, especially in elastic cartilage. Dyscirculatory processes in vessels of own plate of a mucous membrane and a submucous basis of various departments of a larynx were registered. Necrotic changes developed in the columnar epitheliocytes of the submucosal mucosa. Mitochondria swelled, their matrix brightened, cristae collapsed. The results of the article correspond to the research plan of Danylo Halytskyi Lviv National Medical University and are part of the research topic of the Department of Normal Anatomy "Morphofunctional features of organs in pre - and postnatal periods of ontogenesis, under the influence of opioids, food supplements, reconstructive surgery and obesity" 0120U002129) during 2020 - 2024.

Alternative Use of the Insecticide Diofenolan on Musca domestica (Diptera: Muscidae): A Morphological and Ultrastructural Investigation

Musca domestica Linnaeus (Diptera: Muscidae), a relevant synanthropic pest, is the most frequent dipteran species in farms and waste landfills. Insect Growth Regulators (IGR), insecticides with species-specific toxicity and low health and environmental impact, are known to act mostly on larval stages but may induce sterility in adults. We investigated the effects of diofenolan, an IGR analogue of juvenile hormone, on M. domestica, with special attention to female reproductive ability (egg-laying and hatching), and ovarian and ovariole morphology, using optical and transmission electron microscopy. We also tested the interactions between diofenolan and the activity of corpora allata, endocrine gland producing juvenile hormone required for ovarian development, by morphological and ultrastructural investigations. The results show that diofenolan negatively affects the reproductive ability of M. domestica, causing extensive morphological alterations in ovaries and ovarioles. In treated females, ovarioles showed nine different morphotypes that could be arranged into three “transformation paths”, and these alterations were able to reduce egg-laying. The effects of diofenolan on corpora allata, investigated at the optical and ultrastructural level in M. domestica, showed extensive alterations of the nuclei, cytoplasms, and mitochondria, strongly suggesting a rapid transition of the gland from inactivity to degeneration. The sterilizing effects of diofenolan in M. domestica are very interesting for integrated pest management programs within sustainable defence strategies against this relevant and annoying pest.

Dynamics of pathomorphological changes in the structural organization of the intervertebral disc at the end of the seventh and fourteenth day of experimental opioid exposure at the ultrastructural level

In general, the modern literature pays attention to the issues of spine pathology and intervertebral discs. A significant percentage of vertebral disorders - scoliosis, osteochondrosis, spinal disc herniation, etc., occur as a result of exposure to various factors and manifest in changes of the intervertebral discs. The aim of our work was to study at the ultrastructural level the features of pathomorphological manifestations in the structural components of the intervertebral disc at the end of the seventh and fourteenth days of experimental opioid exposure. Materials and methods of research. The objects of the study were 32 sexually mature, white, male rats, weighing 92 - 103 g, aged 4.5 months. Animals were injected with nalbupine intramuscularly once daily (at 10-11 a.m.) for 14 days. The initial dose of nalbuphine was 8 mg/kg during the first week, 15 mg/kg during the second week. It created the conditions of chronic opioid exposure. Before sampling, the animals were withdrawn from the experiment using dibutyl ether. Intervertebral discs of rats were used as a material for ultrastructural study. Ultrastructural specimens were prepared according to the accepted methods. The results of the study. As a result of the sampling after 7 days of opioid exposure we found inhomogeneous osmiophilicity and compaction of the nucleus pulposus matrix in which intensively accumulated osmiophilic grains of glycogen proteoglycans, increased the number of collagen fibers, some of them were heterogeneous. It was also noted the development of moderate degenerative changes in some notochondral cells, which was accompanied by increased vacuolization of the cytoplasm by inhomogeneous compaction of the nucleus and an increase of heterochromatin there. After 14 days necrotic changes in the cells of the nucleus pulposus, as well as the destruction of collagen fibers of the annulus fibrosus were found. In particular, an increase in the amount of heterochromatin in the nucleui of notochondral cells, which was accompanied by a decrease in the volume of the nucleui and inhomogeneous swelling of the cytoplasm. Active fibroblasts were often visualized in the annulus fibrosus. Intense osmiophilicity and thickening of collagen fibers of the annulus fibrosus were observed in some areas of the fibrous ring. Focal destruction of collagen fibers was also noted. In the areas of destruction the fibrils of collagen fibers disintegrated into an inhomogeneous fine-grained stratified mass and were located loosely. Conclusions. At the end of the first week we found that the cytoplasmic processes of chondrocytes decreased in volume, shortened, underwent fragmentation and destruction, some of them detached from the surface of the plasmolemma. At the end of the second week signs of opioid exposure progressed and manifested by an increase in the destruction of cytoplasmic processes in chondrocytes. Also focal destruction of collagen fibers was noted.

Some aspects of the use of oral contraceptives as growth-promoting agents in poultry

There are now several hormone and hormone-like agents that can improve the rate of growth and efficiency of feed use of farm animals. Feeding chickens with oral contraceptive steroids at the dose used by some poultry growers in Egypt has led to the formation of high estrogen residues in the muscles and the liver compared with controls. Electron microscopy of the livers of treated chickens revealed changes at the ultrastructural level. These alterations involve both the nucleus and cytoplasmic organelles, and are similar to those induced by 7, 12 dimethylbenz [a] anthracene in chickens. It is concluded that the use of oral contraceptive steroids in chickens as anabolic agent is a specific risk to chickens and probably to the consumer

Hydrochlorothiazide Use and Risk of Nonmelanoma Skin Cancers: A Biological Plausibility Study

Recent studies reported the association between increased risk of nonmelanoma skin cancers (NMSCs) and the use of hydrochlorothiazide (HCTZ), one of the most commonly prescribed diuretic, antihypertensive drug, over the world. Although HCTZ is known to be photosensitizing, the mechanisms involved in its potential prophotocarcinogenic effects remain unclear. Under acute exposure, therapeutically relevant concentrations of HCTZ (70, 140, and 370 ng/mL) amplified UVA-induced double-strand breaks, oxidative DNA, and protein damage in HaCaT human keratinocytes, and this effect was associated to a defective activity of the DNA repair enzyme, OGG1. Oxidative damage to DNA, but not that to proteins, was reversible within few hours. After chronic, combined exposure to HCTZ (70 ng/mL) and UVA (10 J/cm2), for 9 weeks, keratinocytes acquired a dysplastic-like phenotype characterized by a multilayered morphology and alterations in cell size, shape, and contacts. At the ultrastructural level, several atypical and enlarged nuclei and evident nucleoli were also observed. These transformed keratinocytes were apoptosis resistant, exhibited enhanced clonogenicity capacity, increased DNA damage and inflammation, defective DNA repair ability, and increased expression of the oncogene ΔNp63α and intranuclear β-catenin accumulation (a hallmark of Wnt pathway activation), compared to those treated with UVA alone. None of these molecular, morphological, or functional effects were observed in cells treated with HCTZ alone. All these features resemble in part those of preneoplastic lesions and NMSCs and provide evidence of a biological plausibility for the association among exposure to UVA, use of HCTZ, and increased risk of NMSCs. These results are of translational relevance since we used environmentally relevant UVA doses and tested HCTZ at concentrations that reflect the plasma levels of doses used in clinical practice. This study also highlights that drug safety data should be followed by experimental evaluations to clarify the mechanistic aspects of adverse events.

SARS-CoV-2 infection induces the dedifferentiation of multiciliated cells and impairs mucociliary clearance

Understanding how SARS-CoV-2 spreads within the respiratory tract is important to define the parameters controlling the severity of COVID-19. Here we examine the functional and structural consequences of SARS-CoV-2 infection in a reconstructed human bronchial epithelium model. SARS-CoV-2 replication causes a transient decrease in epithelial barrier function and disruption of tight junctions, though viral particle crossing remains limited. Rather, SARS-CoV-2 replication leads to a rapid loss of the ciliary layer, characterized at the ultrastructural level by axoneme loss and misorientation of remaining basal bodies. Downregulation of the master regulator of ciliogenesis Foxj1 occurs prior to extensive cilia loss, implicating this transcription factor in the dedifferentiation of ciliated cells. Motile cilia function is compromised by SARS-CoV-2 infection, as measured in a mucociliary clearance assay. Epithelial defense mechanisms, including basal cell mobilization and interferon-lambda induction, ramp up only after the initiation of cilia damage. Analysis of SARS-CoV-2 infection in Syrian hamsters further demonstrates the loss of motile cilia in vivo. This study identifies cilia damage as a pathogenic mechanism that could facilitate SARS-CoV-2 spread to the deeper lung parenchyma.