Immunopositive Cells Research Articles

Immunoreactivity of glutamine synthetase in satellite glia around various subpopulations of lumbar dorsal root ganglia neurons in adult rats treated with monosodium glutamate

Satellite glial cells (SGCs), involved inter alia in glutamate (Glu) metabolism, form a glial sheath around sensory neurons of dorsal root ganglia (DRGs). SGCs show a presence of glutamine synthetase (GS) which transform uptaken Glu into glutamine (Gln). In DRGs, this aminoacid is used mainly by small neurons which are able to synthetize substance P (SP) that play a crucial role in nociception. The aim of the study was to define the influence of monosodium glutamate (MSG) on GS immunoreactivity in satellite glia around various subpopulations of neurons including SP immunopositive cells in DRGs of adult rats. The studies were carried out on lumbar DRGs slides in rats which received subcutaneous injection of saline solution (control group) or 4 g/kg b. w. of MSG (MSG group). Immunofluorescence reactions were conducted with use of anti-GS and anti-SP antibodies. Administration of MSG to adult rats increased the GS immunoexpression in SGCs. In rats receiving MSG, a number of small neurons with GS-immunopositive glial sheath was not altered when compared to control individuals, whereas there was a statistically significant increase of GS immunoexpression in SGCs around large and medium neurons. Moreover, in these animals, a statistically significant increase in the number of small SP-positive neurons with GS-positive glial sheath was observed. SP is responsible for transmission of pain, thus the obtained results may be useful for further research concerning the roles of glia in nociceptive pathway regulation.

Osteoblastogenesis and osteolysis in the Zucker Diabetic Sprague Dawley rat humerus head.

The endocrinology of type 2 diabetes (T2D) and its predisposing factors have been studied extensively while its skeletal effects have received negligible research despite this being a global disease. The cellular and molecular association between proximal humeral fractures and T2D has not been fully elucidated. We aimed to study bone cell quantities and immunolabel osteogenic and antiosteogenic cytokines. The study used 12-week-old rats (23 males) consisting of 8 Sprague Dawley (SD) and 15 Zucker Diabetic Sprague Dawley (ZDSD). Weekly mass measurements were taken while fasting blood glucose levels were recorded every 2 weeks with oral glucose tolerance tests conducted once every 4 weeks. Upon termination at the age of 28 weeks, humeri were fixed in 10% buffered formalin, prior to decalcification in ethylenediaminetetraacetic acid. The bone samples were then processed in ascending grades of alcohol using an automatic processor before embedding in paraffin wax. Sections were cut at 5 µm thickness in a series for Haematoxylin and Eosin stain, and immunohistochemistry was performed with the anti-tartrate-resistant acid phosphatase (TRAP), anti-alkaline phosphatase (ALP), anti-bone morphogenetic protein 3 (BMP3), anti-transforming growth factor beta 1 (TGFβ1), anti-aged glycation end product (AGE) antibodies in the sequence. ZDSD rats had more adipocytes, BMP3 and AGEs expression with higher numbers of TRAP positive osteocytes and fewer ALP cells although no differences were found in TGFβ1 immunopositivity. We also found that T2D increases the number of AGEs immuno-positive cells, as well as its extracellular expression, thus providing a conducive environment for the interaction of the osteogenic cytokine and its antagonist to suppress osteoblastogenesis. ZDSD groups had higher adipocyte numbers therefore increased marrow adiposity in T2D.

Stress effects on spatial memory retrieval and brain c-Fos expression pattern in adults are modulated by early nicotine exposure

The cognitive effects of nicotine are linked to persistent modifications in extended neural systems that regulate cognitive and emotional processes, and these changes occur during development. Additionally, acute stress has modulatory effects on cognition that involve broad neural systems and can be influenced by prior environmental challenges. The effects of nicotine and stress may be interconnected, leading to modifications in a network of shared brain substrates. Here, we explored the interaction between nicotine and stress by evaluating the effects of acute stress exposure in spatial memory retrieval for animals pretreated with nicotine during adolescence or adulthood. Adolescent (35 days old) and adult (70 days old) male Wistar rats were treated for 21 days with one daily subcutaneous injection of nicotine 0.14 mg/ml (free base). 30 days after the last injection, rats were trained in the Barnes maze and tested 24 h later, half the rats were tested under regular conditions, and half of them were exposed to 1 h of restraining stress before the retrieval test, and brain samples were collected and c-Fos immunopositive cells were stained. Prolonged nicotine withdrawal or acute stress improved spatial memory retrieval. Acute stress in nicotine pretreated adults impaired spatial memory retrieval. Nicotine exposure during early adulthood resulted in long-lasting brain adaptations that amplified emotional responses to acute stress after prolonged drug withdrawal.

A solid lipid particle formulation of long pepper extract reduces pain and astrocyte activation in a rat model of neuropathic pain.

To investigate the effects of solid lipid microparticle (SLM) creams containing a long pepper extract (LPE) or piperine on neuropathy-related pain and the expression of Glial fibrillary acidic protein (GFAP) as a measure of astrogliosis. Neuropathic pain in male Spraque Dawley rats was induced by sciatic nerve ligation (SNL) and followed by treatment with LPE-SLM, piperine-SLM, capsaicin or vehicle creams. The pain score was assessed by thermal hyperalgesia test. The GFAP expression in the spinal cord was determined by immunohistochemistry. Pain scores were significantly increased after SNL and decreased when treated by LPE-SLM. The number of GFAP immunopositive cells was significantly increased in the SNL rats. Treated by LPE-SLM and capsaicin creams resulted in a significant reduction of the number of GFAP immunopositive cells. The LPE-SLM treated rats showed greater effects than the piperine and capsaicin preparations. The LPE-SLM cream has a potential effect on pain attenuation via a decrease of spinal astrocyte activation-related mechanism. The LPE in SLM preparation could provide an alternative therapeutic strategy for treating neuropathic pain.

The role of inflammation and antioxidant defenses in the cardiotoxicity of doxorubicin in elderly CD-1 male mice

Doxorubicin (DOX) is a potent chemotherapeutic agent used against several cancer types. However, due to its cardiotoxic adverse effects, the use of this drug may be also life-threatening. Although most cancer patients are elderly, they are poorly represented and evaluated in pre-clinical and clinical studies. Considering this, the present work aims to evaluate inflammation and oxidative stress as the main mechanisms of DOX-induced cardiotoxicity, in an innovative approach using an experimental model constituted of elderly animals treated with a clinically relevant human cumulative dose of DOX. Elderly (18–20 months) CD-1 male mice received biweekly DOX administrations, for 3 weeks, to reach a cumulative dose of 9.0 mg/kg. One week (1W) or two months (2 M) after the last DOX administration, the heart was collected to determine both drug’s short and longer cardiac adverse effects. The obtained results showed that DOX causes cardiac histological damage and fibrosis at both time points. In the 1W-DOX group, the number of nuclear factor kappa B (NF-κB) p65 immunopositive cells increased and a trend toward increased NF-κB p65 expression was seen. An increase of inducible nitric oxide synthase (iNOS) and interleukin (IL)-33 and a trend toward increased IL-6 and B-cell lymphoma-2-associated X (Bax) expression were seen after DOX. In the same group, a decrease in IL-1β, p62, and microtubule-associated protein 1A/1B-light chain 3 (LC3)-I, p38 mitogen-activated protein kinase (MAPK) expression was observed. Contrariwise, the animals sacrificed 2 M after DOX showed a significant increase in glutathione peroxidase 1 and Bax expression with persistent cardiac damage and fibrosis, while carbonylated proteins, erythroid-2-related factor 2 (Nrf2), NF-κB p65, myeloperoxidase, LC3-I, and LC3-II expression decreased. In conclusion, our study demonstrated that in an elderly mouse population, DOX induces cardiac inflammation, autophagy, and apoptosis in the heart in the short term. When kept for a longer period, oxidative-stress-linked pathways remained altered, as well as autophagy markers and tissue damage after DOX treatment, emphasizing the need for continuous post-treatment cardiac monitoring.

Expression of Fascin and SALL4 in odontogenic cysts and tumors: an immunohistochemical appraisal.

Background: Various stemness markers (SOX2, OCT4, and NANOG) have been studied in odontogenic cysts and tumors. However, studies on SALL4 having similar properties of stemness has not been documented. Additionally, insight into fascin as a migratory molecule is less explored. Following a thorough literature search we hypothesize that fascin might contribute for the local migratory behaviour of the odontogenic epithelial cells in tumors and cysts while SALL4 may contribute to stemness property. Thus, the aim of the present study was to evaluate the expression of fascin and SALL4 in histopathological variants of Ameloblastoma, Adenomatoid Odontogenic Tumor (AOT) and various odontogenic cysts namely Odontogenic keratocyst (OKC), Dentigerous cyst (DC), Radicular cyst (RC), and Calcifying odontogenic cyst (COC). Methods: Semi-quantitative analysis of fascin and SALL4 immuno-positive cells was evaluated in a total of 40 cases of ameloblastoma (11 plexiform, 12 follicular, 12 unicystic, and 5 desmoplastic) variants, 6 cases of AOT, 15 cases each of OKC, DC, RC and 5 of COC. Chi-square test was applied to evaluate the association between SALL4 and fascin expression in odontogenic cysts and tumors. Results: Fascin immunopositivity was observed in peripheral ameloblast-like cells, and the expression was weak or absent in stellate reticulum-like cells. A moderate to weak immune-reactivity to SALL4 was observed in the cytoplasm of ameloblastoma, epithelial cells of DC and RC having a marked inflammatory infiltrate, which was an interesting observation. COC and AOT had negative to weak expressions. Conclusion: Expression of fascin in ameloblastomas elucidate their role in motility and localized invasion. Its expression in less aggressive lesions like DC, COC, AOT will incite to explore the other functional properties of fascin. SALL4 expression in the cytoplasm of odontogenic cysts and tumors may represent inactive or mutant forms which requires further validation.

The protective effect of Aspalathus linearis against the experimental gastric ulcer induced by ethanol in rats

This study aimed to investigate the effect of Aspalathus linearis on the expression levels of major histocompatibility complex (MHC) molecules and their ability to present antigens to CD8, CD68, and T cells in an experimental rat model of gastritis. Furthermore, changes in tumor necrosis factor–alpha (TNF–α), myeloperoxidase, superoxide dismutase, and malondialdehyde levels due to the antioxidant properties of the plant were evaluated. The study included 24 female Wistar albino rats (Rattus norvegicus). A. linearis was administered to the treatment groups of an ethanol–induced gastritis rat model via oral gavage for 5 days the blood and stomach tissues were sampled for analysis. Biochemical evaluation revealed that TNF–α and myeloperoxidase levels decreased and superoxide dismutase activity increased in the A. linearis–treated groups compared with the ethanol–treated group. Histochemical evaluation revealed that the severity of inflammation decreased in the rats with gastritis treated with A. linearis. In addition, the number of cells expressing CD8 and CD68 decreased. The number of MHC–II immunopositive cells was higher in the A. linearis–treated group compared with the control and other treatment groups. A. linearis exhibits a protective effect on gastric mucosa by reducing the severity of oxidative stress owing to its antioxidant, anti–inflammatory, and antiapopitotic properties.

Investigation of Effects of Local Vitamin C Application on Inflammatory Response and Periodontal Tissue Destruction in Rat Periodontitis Model

Background: The present study aims to evaluate the effect of local vitamin C application on inflammatory response and periodontal tissue destruction in rats with experimental periodontitis.
 Methods: A total of 21 animals, 7 rats in each group, were used in the study: 1- Non-ligation (NL), 2-experimental periodontitis (EP), and 3- local vitamin C (VtC) groups. Experimental periodontitis was induced in EP and VtC group rats. After 11 days, the ligature was removed. 50 mL of saline solution and vitamin C were injected locally into the vestibule sulcus of the EP and VtC group rats, respectively. After the rats were sacrificed, blood and gingival tissue samples were taken. TNF-α levels were analyzed biochemically in serum and 8-OHdG and MMP-8 were analyzed immunohistochemically in gingival tissue. The mandibular specimens were histologically evaluated for bone destruction and attachment loss.
 Results: The numeric density of 8-OHdG and MMP-8 immunopositive cells were found to be statistically significantly lower in the Vtc group than in the EP group. Vitamin C administration significantly reduced attachment loss and bone loss in the VtC group as compared to the EP group. However, there was no significant difference in serum TNF-α level in the VtC group compared to the EP group.
 Conclusions: Local application of vitamin C may reduce inflammation-related bone destruction by supporting the inflammatory response with the antioxidant activity of vitamin C and contributing to the resolution of inflammation with its potential immunomodulatory effect. Vitamin C may be a therapeutic agent that can be used in the treatment of periodontitis.

Innate and adaptive immune gene expression in the brain is associated with neuropathological changes after infection with bovine alpha-herpesvirus-5 in mice

Bovine alpha herpesvirus-5 (BoAHV-5) is related to the development of meningoencephalitis in cattle. Very little is known about the molecular pathways involved in the central nervous system (CNS) damage associated with inflammation during BoHV-5 infection in mice. To better identify the specific immunological pathways triggered by BoAHV-5 infection in mice, we evaluated the mRNA expression of 84 genes involved in innate and adaptive immune responses. We compared gene expression changes in the cerebrum from noninfected and infected mice with BoHV-5 at a 1 × 107 TCID50. Then, we analyzed the association of these genes with neurological signs, neuropathology, and activation of glial cells in response to BoHV-5 infection. Three days after BoAHV-5 infection, increased expression of TNF, IL-2, CXCL10, CXCR3, CCR4, CCL5, IFN-γ, IL-10, IRF7, STAT1, MX1, GATA 3 C3, LIZ2, caspase-1 and IL-1b was found. We also observed the upregulated expression of the CD8a, TBX21 and CD40LG genes and the downregulated expression of the CD4 gene after BoAHV-5 infection. In addition, BoHV-5-infected animals showed higher levels of all the evaluated inflammatory mediators (TNF, IFN-γ and IL-10) on day 3 postinfection. BoAHV-5-infected animals showed neurological changes along with meningoencephalitis, neuropil vacuolation, hemorrhage and reactive gliosis. Astrogliosis and microgliosis, indicated by increased expression of glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (Iba-1), were found throughout the neuropil in infected brains. Moreover, cleaved caspase-3 immunopositive glio-inflammatory cells were visualized around some blood vessels in areas of neuroinflammation in the cerebrum. In agreement on that we found higher cleaved caspase-3 and Iba-1 expression evaluated by western blot analysis in the brains of infected mice compared to control mice. In conclusion, our results revealed

FEATURES OF PROLIFERATION AND APOPTOSIS OF COLONIC EPITHELIOCYTES AFTER ELECTRON IRRADIATION AT A DOSE OF 8 GY

Intestinal epithelial cells are characterized by high mitotic activity and have high radiosensitivity to all types of ionizing radiation that cause their damage. In modern morphology and radiology, there is no data on the effect of active electrons on the intestinal mucosa, despite the promise and active implementation of this type of radiation for therapeutic purposes in oncology. Purpose of the study: immunohistochemical assessment of proliferation and apoptosis of colon epithelial cells in male Wistar rats after local irradiation with electrons at a dose of 8 Gy. Animals were irradiated using a linear accelerator NOVAC-11, at a dose rate of 1 Gy/min, energy of 10 MeV and frequency of 9 Hz, the diameter of the irradiation field of the pelvic-abdominal region was 100 mm. Animals from each group were removed from the experiment on the first and third days. Histological preparations of the colon (n=30) from two groups, the first control group (n=10) and the second experimental group (n=20), were examined by immunohistochemical method with antibodies to Ki-67 and Cas3. In preparations of rats bred one day after irradiation, a predominance of apoptotic processes was revealed. The number of positive cells stained with antibodies to caspase 3 was increased by 1.5 times in the experimental group compared to the control group, and the number of Ki-67-positive cells, on the contrary, was reduced by 1.5 times. On day 3 after electron irradiation in the epithelium of the colon, the number of immunopositive cells did not differ from control values. Thus, with local single irradiation of the pelvic-abdominal region of rats with electrons at a dose of 8 Gy, a decrease in the number of stem epithelial cells of the intestinal crypts of the colon is observed every other day, which is confirmed by a shift in the proliferative-apoptotic balance towards cell death. In the dynamics of observation on the third day of the experiment, an increase in the pool of proliferating cells is observed, followed by restoration of intestinal crypts.

Audiogenic Seizures in the Streptozotocin-Induced Rat Alzheimer's Disease Model.

Alzheimer's disease (AD) is a neurodegenerative and progressive disorder with no cure and constant failures in clinical trials. The main AD hallmarks are amyloid-β (Aβ) plaques, neurofibrillary tangles, and neurodegeneration. However, many other events have been implicated in AD pathogenesis. Epilepsy is a common comorbidity of AD and there is important evidence indicating a bidirectional link between these two disorders. Some studies suggest that disturbed insulin signaling might play an important role in this connection. To understand the effects of neuronal insulin resistance in the AD-epilepsy link. We submitted the streptozotocin (STZ) induced rat AD Model (icv-STZ AD) to an acute acoustic stimulus (AS), a known trigger of seizures. We also assessed animals' performance in the memory test, the Morris water maze and the neuronal activity (c-Fos protein) induced by a single audiogenic seizure in regions that express high levels of insulin receptors. We identified significant memory impairment and seizures in 71.43% of all icv-STZ/AS rats, in contrast to 22.22% of the vehicle group. After seizures, icv-STZ/AS rats presented higher number of c-Fos immunopositive cells in hippocampal, cortical, and hypothalamic regions. STZ may facilitate seizure generation and propagation by impairment of neuronal function, especially in regions that express high levels of insulin receptors. The data presented here indicate that the icv-STZ AD model might have implications not only for AD, but also for epilepsy. Finally, impaired insulin signaling might be one of the mechanisms by which AD presents a bidirectional connection to epilepsy.

Poster 120: Understanding the Biological Basis of Contracture Using a Mouse Model of Single Hindlimb Immobilization-Induced Fibrosis

Poster 120: Understanding the Biological Basis of Contracture Using a Mouse Model of Single Hindlimb Immobilization-Induced Fibrosis

DISTRIBUTION OF PARVALBUMIN-EXPRESSING NEURONAL POPULATIONS IN THE CAT CERVICAL AND LUMBAR SPINAL CORD GRAY MATTER

Parvalbumin is a classical marker of interneuronal populations in the central nervous system. Analyzing the cervical and lumbar spinal cord segments of cats (Felis catus), both individual cells and entire populations of neurons expressing parvalbumin were identified in most of the gray matter laminae. These populations have strict laminar and nuclear localization. Numerous neuronal clusters are located in the medial part of lamina V–VI and in laminae VII of cervical and lumbar enlargements. We believe that the first one located in segments C4–C8 and L4–L7 may participate in the modulatory mechanisms of locomotor activity via the convergence of cutaneous and proprioceptive afferentation from the limbs. Neuronal populations in lamina VII consist of Ia interneurons and Renshaw interneurons that participate in the motoneuron inhibition. Less numerous populations of parvalbumin-immunopositive cells found in laminae III possibly participated in the regulation of cutaneous sensitivity. Another population located in lamina VIII possibly forms commissural and propriospinal connections and participates in modulating the activity of motoneurons. Immunopositive interneurons also revealed in the precerebellar nuclei: central cervical nucleus and Clarke’s nucleus; unlike the general population of these nuclei, neurons revealed are interneurons. Scarce immunopositive cells are found in lamina I of L6–L7 segments, as well as in laminae II, IV, and X of all segments investigated.

Immunohistochemical analyses of neural stem cell lineage markers in normal feline brains and glial tumors.

Neural stem cell (NSC) lineage cells have not been fully identified in feline brains, and the NSC-like nature of feline glial tumors has not been determined. In this study, 6 normal cat brains (3 newborn and 3 older cats) and 13 feline glial tumors were analyzed using immunohistochemical NSC lineage markers. The feline glial tumors were subjected to immunohistochemical scoring followed by hierarchical cluster analysis. In newborn brains, glial acidic fibrillary protein (GFAP)/nestin/sex-determining region Y-box transcription factor 2 (SOX2)-immunopositive NSCs, SOX2-immunopositive intermediate progenitor cells, oligodendrocyte transcription factor 2 (OLIG2)/platelet-derived growth factor receptor-α (PDGFR-α)-immunopositive oligodendrocyte precursor cells (OPCs), OLIG2/GFAP-immunopositive immature astrocytes, and neuronal nuclear (NeuN)/β-3 tubulin-immunopositive mature neuronal cells were observed. The apical membrane of NSCs was also immunopositive for Na+/H+ exchanger regulatory factor 1 (NHERF1). In mature brains, the NSC lineage cells were similar to those of the newborn brains. A total of 13 glial tumors consisted of 2 oligodendrogliomas, 4 astrocytomas, 3 subependymomas, and 4 ependymomas. Astrocytomas, subependymomas, and ependymomas were immunopositive for GFAP, nestin, and SOX2. Subependymomas and ependymomas showed dot-like or apical membrane immunolabeling for NHERF1, respectively. Astrocytomas were immunopositive for OLIG2. Oligodendrogliomas and subependymomas were immunopositive for OLIG2 and PDGFR-α. Feline glial tumors also showed variable immunolabeling for β-3 tubulin, NeuN, and synaptophysin. Based on these results, feline astrocytomas, subependymomas, and ependymomas appear to have an NSC-like immunophenotype. In addition, astrocytomas, subependymomas, and ependymomas have the characteristics of glial, oligodendrocyte precursor, and ependymal cells, respectively. Feline oligodendrogliomas likely have an OPC-like immunophenotype. In addition, feline glial tumors may have multipotential stemness for differentiation into neuronal cells. These preliminary results should be validated by gene expression analyses in future studies with larger case numbers.

Characterization and co-localization of thyroid stimulating hormone and melatonin receptors in the pars tuberalis of mares during the breeding and non-breeding seasons

Control of seasonal reproduction in horses has yet to be fully elucidated; however, duration of melatonin secretion is a seasonal regulator of the hypothalamic-pituitary-gonadal (HPG) axis. The hypophyseal pars tuberalis (PT) appears to play a key role in the transduction of melatonin signal through melatonin responsive, PT-specific cells that produce thyroid stimulating hormone (TSH). The objectives of these experiments were to characterize and colocalize TSH-producing cells and melatonin receptors (MT1r) in the PT during breeding and non-breeding seasons. Pituitaries from nine light-horse type mares, aged 6 to 26 years, were obtained following euthanasia during the breeding and non-breeding seasons. Mares were determined to be either cycling (n = 5) or seasonally anovulatory (n = 4) based on season, gross inspection of ovarian structures, and plasma progesterone concentrations. Immediately following euthanasia, the carotid artery was perfused with sodium nitrite and 4% paraformaldehyde before decapitation and dissection of PT from the hypophysis. Immunofluorescent detection of TSH was carried out on fixed-frozen PT sections and number of immunopositive cells per 1 mm2 were counted and averaged for three replications per horse to compare TSH distribution between breeding and non-breeding season using Mann Whitney U test. TSH-immunoreactive cells were present in PT and were greater (P < 0.05) in PT during the breeding season comparedto non-breeding season; few to no immunoreactive cells were seen in PT from non-breeding season. In situ hybridization (ISH) was carried out using RNAscope HD Red Detection Kit and a custom MT1r probe (Ec-MTNR1A) to confirm MT1r localization and compare MT1r distribution between breeding and non-breeding seasons. Mean percentage of MT1r immunopositive cells, determined as a percentage of total number of cells, and expression of MT1r mRNA per cell, determined as mean number of dots (representing mRNA) per cell, were analyzed using Mann Whitney U test. Melatonin receptor mRNA was densely expressed in glandular cells of the PT and no differences were found in either percent immunopositive cells or mean number of mRNA dots per cell between breeding and non-breeding seasons. To determine if MT1r colocalize with TSH-producing cells, ISH was carried out alongside immunofluorescent detection of TSH as previously described. In cycling mares, MT1r mRNA colocalized with TSH-producing cells in the PT; however, not all TSH-producing cells colocalized with MT1r and vice versa. Lack of TSH-producing cells accounted for absence of colocalization in seasonally anovulatory mares. The PT is considered a major target for melatonin, and colocalization with TSH supports a role for TSH as a modulator of seasonal reproduction in the mare. This is further evidenced by increased TSH immunosignal during the breeding season.

Morphological and biochemical evaluation of effects of Myrtus communis L. extract on heart and aorta in high fat-diet-induced obese rats

Objective: The purpose of this study was to examine the protective effects of Myrtus communis L. (MC) extract on high fat-diet (HFD) induced heart and aorta damage by evaluating oxidative stress and the endothelial nitric oxide system (eNOS). Materials and Methods: Wistar albino male rats were divided into 3 groups (n=7) as control, HFD, and HFD+MC. Rats in HFD and HFD+MC groups were HFD fed for 16 weeks and in the last 4 weeks saline or MC (100 mg/kg) was administered orally (5 days/week). Triglyceride, cholesterol, and high-density lipoprotein (HDL) were estimated in blood serum. Tissue oxidative stress and inflammatory parameters were evaluated biochemically. Tissue morphologies, eNOS, inducible NOS (iNOS), and NADPH oxidase-2 (NOX-2)-immunopositive and apoptotic cells were evaluated histologically. Results: Altered serum lipid profiles, degenerated heart, and aorta morphology, increased malondialdehyde, 8‐hydroxy‐2‐ deoxyguanosine, tumor necrosis factor-alpha, monocyte chemoattractant protein-1 and myeloperoxidase levels, and iNOS, NOX-2 immunopositive and apoptotic cells, decreased NO levels, eNOS-immunopositive cells in both tissues were observed in HFD group. All these parameters improved in the HFD+MC group. Conclusion: This study revealed that HFD-induced obesity increased iNOS activation and oxidative stress in the cardiac and aortic tissues of the rats. MC improved oxidant/antioxidant balance and prevented heart and aorta damage via eNOS involvement.

P53 expression is associated with tumor stage, grade and subtype in patients with hepatocellular carcinoma.

The present study aimed to determine the expression levels of p53 in patients with hepatocellular carcinoma (HCC) and to evaluate its association with several HCC-related prognostic factors and in particular, with tumor stage, grade and subtype. Therefore, a cross-sectional study, involving 41 patients with HCC, who underwent surgical resection between January, 2013 and December, 2020 was conducted. To assess the expression levels of p53 in all patients with HCC, immunohistochemical staining was performed. In addition, the association between p53 expression and the clinicopathological characteristics of patients with HCC, including prognostic factors, was evaluated by applying the appropriate statistical analysis methods. The results revealed that among the 41 patients enrolled, 35 patients (85.4%) were positive for p53 expression. A higher percentage of positive p53 expression was observed in male patients >60 years old, with single HCC nodules >5 cm in diameter and vascular invasion, compared with their counterparts. A positive p53 expression was associated with well- and poorly differentiated HCC, but not with tumor stage and subtype. No differences in p53 expression were observed across different tumor stages and subtypes. Additionally, patients with moderately and poorly differentiated HCC exhibited significantly higher p53 expression levels compared with those suffering from well-differentiated HCC. Overall, the results demonstrated that the rate of p53 immuno-positive cells was increased in patients with HCC. In addition, p53 expression was associated with well- and poorly differentiated HCC, thus suggesting its association with a poorer prognosis.

(132) Histometric Measurement of CD117 and PGP9.5 Immunopositive Staining in Excised Vestibular Specimens from Patients with Neuroproliferative Vestibulodynia

Abstract Introduction In 2004, Bornstein reported using immunohistochemical (IHC) analysis of stained sections of vestibular specimens, defining diagnostic criteria for neuroproliferative vestibulodynia (NPV). Criteria included presence of 8 or more mast cells manually counted in a standardized microscopic field or finding total area of subepithelial nerve fibers was 10 times higher than controls. Replicating such criteria is difficult, in part, because of variability in manual counting, IHC staining and magnification. A more reproducible methodology may be estimating percentage of immunopositive stained area using computer-assisted histometry. Objective Utilizing both manual counting and computer-assisted histometry measurements using ImageJ at a standardized magnification in specimens from vestibulectomy and control tissue published in the literature. Methods Tissue samples were collected from 17 patients with NPV. Samples were stained with CD117, a marker of mast cells, and PGP9.5, a marker of nerve endings. Twenty-five photomicrographs were each obtained using 200x and 400x magnification and all were analyzed using ImageJ. Images were converted to 8-bit grayscale and threshold manually adjusted to measure immunohistochemically stained tissue with three measurements averaged. Averages and variability between measurements in each photomicrograph and from each patient were determined. This methodology was also applied to images of tissues from patients with and without NPV (Tympanidis 2003; Goetsch 2010). For comparison, the method of analysis by manually counting CD117 immunopositive cells, described by Bornstein et al, was applied to our tissue samples by an independent observer. Clusters of immunopositive granules positively stained and separated from an adjacent cell membrane were counted as a single immunopositive cell. Results Using Image J, tissues stained with CD117 averaged 2107 ± 1339 μm2 of immunostained area, representing 0.68 ± 0.38% (range = 0.11 – 1.15%) of total area analyzed. Tissues stained with PGP9.5 averaged 1123 ± 695 μm2 of immunostained area, representing 0.40 ± 0.27% (range = 0.34 – 1.81%) of total area analyzed. Standard deviations of repeated measurements for the same area within a tissue section ranged from 0.04 – 0.7% (mean = 0.17%) for CD117 immunostaining and 0.01 – 0.78% (mean = 0.10%) for PGP 9.5. Counting cells, CD117 stained tissue averaged 30.6 ± 15.3 (range = 12 – 72) immunopositive cells per standardized field (200x magnification). These are all greater than the 8 mast cells per standardized field reported by Bornstein. In PGP9.5-stained tissue, immunostained area was 2.6-fold greater than that reported by Bornstein for controls (425 μm2). Applying our methodology to photomicrographs from Goetsch and Tympanidis, 2.48% and 2.87% of the control areas were immunopositive. Tissues from control patients without vestibulodynia had 8-fold lower (0.31%, from Goetsch) and 6.4-fold lower (0.45%, from Tympanidis) immunostained area than tissues from vestibulodynia patients using computer-assisted histometry. Conclusions NPV is a diagnosis of exclusion without routine pathology confirmation. Our 17 patients had increased density of CD117-immunopositive cells and increased PGP9.5-immunopositive nerves, confirming their NPV diagnosis. To facilitate more consistent, widespread and rapid quantification of staining, we propose using computer-assisted histometry for future studies. Disclosure No

Orexin receptors in paraventricular nucleus influence sexual behavior via regulating the sympathetic outflow in males.

Orexins are hypothalamic neuropeptides associated with various neurophysiological activities such as sleep, arousal, and reward. However, there are few studies investigating the relationships between orexin receptors in the paraventricular nucleus and sexual behaviors. To explore the roles of orexin receptors in the paraventricular nucleus on sexual behaviors and uncover its potential mechanisms in males. Orexin A, orexin 1 receptor antagonist SB334867, and orexin 2 receptor antagonist TCS-OX2-29 were microinjected into the paraventricular nucleus to investigate the effects of orexin receptors on copulatory behavior testing of C57BL/6 mice. To explore if ejaculation could activate orexin 1 receptor-expressing neurons in the paraventricular nucleus, fluorescence immunohistochemical double staining was utilized. The levels of serum norepinephrine were measured and the lumbar sympathetic nerve activity was recorded to reflect the sympathetic nervous system activity. Moreover, the bulbospongiosus muscle-electromyogram was recorded and analyzed. To test whether perifornical/lateral hypothalamic area orexinergic neurons directly projected to the paraventricular nucleus, virus retrograde tracing technology was utilized. Orexin A significantly enhanced sexual performance by shortening the intromission and ejaculation latencies, and increasing the mount and intromission frequencies, while the opposite outcomes appeared with SB334867. However, TCS-OX2-29 had no significant effects on sexual behaviors. Moreover, orexin A increased lumbar sympathetic nerve activity and the levels of serum norepinephrine, while SB334867 decreased lumbar sympathetic nerve activity and norepinephrine, which caused a significant decrease in sympathetic nervous system outflow. Meanwhile, a robust increase in the bulbospongiosus muscle-electromyogram activity was identified after microinjecting orexin A. Furthermore, cFos immunopositive cells were increased and double stained with orexin 1 receptor-expressing neurons in the mating group. Additionally, the retrograde tracing results demonstrated that orexinergic neurons in the perifornical/lateral hypothalamic area directly projected to the paraventricular nucleus. Orexin 1 receptor in the paraventricular nucleus could influence the ejaculatory reflex via mediating the sympathetic nervous system activity, which might be of great importance in the treatment of premature ejaculation in the future.

MP27-15 A COMPARISON OF COMPUTER-ASSISTED ANALYSES AND MANUAL COUNTING IN VESTIBULAR TISSUE STAINED WITH PGP9.5 AND CD117

You have accessJournal of UrologyCME1 Apr 2023MP27-15 A COMPARISON OF COMPUTER-ASSISTED ANALYSES AND MANUAL COUNTING IN VESTIBULAR TISSUE STAINED WITH PGP9.5 AND CD117 Alexandra Drian, Noel N. Kim, Sue W. Goldstein, Andrew S Goldstein, Alyssa Yee, and Irwin Goldstein Alexandra DrianAlexandra Drian More articles by this author , Noel N. KimNoel N. Kim More articles by this author , Sue W. GoldsteinSue W. Goldstein More articles by this author , Andrew S GoldsteinAndrew S Goldstein More articles by this author , Alyssa YeeAlyssa Yee More articles by this author , and Irwin GoldsteinIrwin Goldstein More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003255.15AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Immunohistochemical (IHC) analysis of stained sections of vestibular specimens have been used to define diagnostic criteria for neuroproliferative vestibulodynia (NPV). Bornstein’s criteria (2004) included manually counting 8 or more mast cells in a standardized microscopic field and/or subepithelial nerve fibers that were 10 times higher than controls. However, there is wide variability in manual counting, IHC staining and magnification. We examined a more reproducible methodology by assessing percentage of immunopositive stained area using computer-assisted histometry. METHODS: Samples from 17 patients were stained with CD117, a marker of mast cells, and PGP9.5, a neuronal marker. Photomicrographs were obtained using 200x and 400x magnification and analyzed using Image J with 3 measurements per photomicrograph. This methodology was also applied to previously published images of tissues from patients with and without NPV (Tympanidis 2003; Goetsch 2010). For comparison, CD117-immunopositive cells were also manually counted, as described by Bornstein. Clusters of immunopositive granules positively stained and separated from an adjacent cell membrane were counted as a single immunopositive cell. RESULTS: Using Image J, mean CD117-immunopositive area was 2107 ± 1339 μm2, representing 0.79 ± 0.51% of total area analyzed. Mean PGP9.5-immunopositive area was 1123 ± 695 μm2, representing 0.38 ± 0.27% of total area analyzed. Using manual counting, we observed 30.6 ± 15.3 CD117-immunopositive cells per standardized field (200x), greater than the Bornstein threshold of 8 mast cells per standardized field. Tissues from control patients without vestibulodynia had 6 to 8-fold lower immunostained area than tissues from vestibulodynia patients using computer-assisted histometry. In analyses of photomicrographs from Goetsch and Tympanidis, 2.48% and 2.87% of the control areas were immunopositive. For controls, using Image J, immunostained area for PGP 9.5 was 2.6-fold greater than that reported by Bornstein using manual counting (425 μm2). CONCLUSIONS: All our patients had increased density of CD117-immunopositive cells and increased PGP9.5-immunopositive area, confirming their NPV diagnosis. To facilitate more consistent, widespread and rapid assessment of IHC analysis of stained sections of vestibular specimens, we propose using computer-assisted histometry for future studies. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e368 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Alexandra Drian More articles by this author Noel N. Kim More articles by this author Sue W. Goldstein More articles by this author Andrew S Goldstein More articles by this author Alyssa Yee More articles by this author Irwin Goldstein More articles by this author Expand All Advertisement PDF downloadLoading ...