Giemsa Research Articles

Effectiveness of May Grunwald Giemsa staining on tissues fixed with sugar cane molasses

Formaldehyde is a reactive chemical that reacts randomly with various cellular elements. This substance, which is also found in the natural structure of the organism, is used in many areas, from the industry to household materials, from the production of coatings in dentistry to the fixation of cadavers in laboratories. Formaldehyde is commonly used in laboratories for fixation of tissues. In this study, the staining properties of tissues fixed with low-cost sugar cane molasses, which is one of the natural sugars and organically produced in Turkey, shown by May Grunwald Giemsa staining method were compared with those of tissues fixed with buffered formol-saline. As a result, intense maceration was observed in the skin and small intestines. Considering the chromatin distribution, nucleus separation and cytoplasm staining in other tissues (liver, spleen, kidney, testis, large intestine, brain, cerebellum and lung), tissues fixed with sugarcane molasses showed similar properties to tissues fixed with buffered formal-saline.

Harmine promotes megakaryocyte differentiation and thrombopoiesis by activating the Rac1/Cdc42/JNK pathway through a potential target of 5-HTR2A.

Harmine (HM), a β-carboline alkaloid extracted from plants, is a crucial component of traditional Chinese medicine (TCM) known for its diverse pharmacological activities. Thrombocytopenia, a common and challenging hematological disorder, often coexists with serious illnesses. Previous research has shown a correlation between HM and thrombocytopenia, but the mechanism needs further elucidation. The aim of this study was to clarify the mechanisms underlying the effects of HM on thrombocytopenia and to develop new therapeutic strategies. Flow cytometry, Giemsa staining, and Phalloidin staining were used to assess HM's impact on Meg-01 and HEL cell differentiation and maturation in vitro. A radiation-induced thrombocytopenic mouse model was employed to evaluate HM's effect on platelet production in vivo. Network pharmacology, molecular docking, and protein blotting were utilized to investigate HM's targets and mechanisms. The results demonstrated that HM dose-dependently promoted Meg-01 and HEL cell differentiation and maturation in vitro and restored platelet levels in irradiated mice in vivo. Subsequently, HM was found to be involved in the biological process of platelet production by upregulating the expressions of Rac1, Cdc42, JNK, and 5-HTR2A. Furthermore, the targeting of HM to 5-HTR2A and its correlation with downstream Rac1/Cdc42/JNK were also confirmed. In conclusion, HM regulates megakaryocyte differentiation and thrombopoiesis through the 5-HTR2A and Rac1/Cdc42/JNK pathways, providing a potential treatment strategy for thrombocytopenia.

Protective Effect of Hibifolin on Lipopolysaccharide-Induced Acute Lung Injury Through Akt Phosphorylation and NFκB Pathway.

Acute lung injury (ALI) is a difficult condition to manage, especially when it is complicated by bacterial sepsis. Hibifolin, a flavonoid glycoside, has anti-inflammatory properties that make it a potential treatment for ALI. However, more research is needed to determine its effectiveness in LPS-induced ALI. In this study, male ICR mice were treated with hibifolin before LPS-induced ALI. Protein content and neutrophil count in bronchoalveolar lavage (BAL) fluid were measured by BCA assay and Giemsa staining method, respectively. The levels of proinflammatory cytokines and adhesive molecules were detected by ELISA assay. The expression of NFκB p65 phosphorylation, IκB degradation, and Akt phosphorylation was assessed by western blot assay. Hibifolin pre-treatment significantly reduced pulmonary vascular barrier dysfunction and neutrophil infiltration into the BAL fluid in LPS-induced ALI mice. In addition, LPS-induced expression of proinflammatory cytokines (IL-1β, IL-6, TNF-α) and adhesive molecules (ICAM-1, VCAM-1) within the BAL fluid were markedly reduced by hibifolin in LPS-induced ALI mice. More, hibifolin inhibited LPS-induced phosphorylation of NFκB p65, degradation of IκB, and phosphorylation of Akt in lungs with ALI mice. In conclusion, hibifolin shows promise in improving the pathophysiological features and proinflammatory responses of LPS-induced ALI in mice through the NFκB pathway and its upstream factor, Akt phosphorylation.

The potential neuroprotective effects of Spirulina platensis in a valproic acid-induced experimental model of autism in the siblings of albino rats: targeting PIk3/AKT/mTOR signalling pathway

ABSTRACT Introduction: Autism spectrum disorders (ASDs) are a group of neurodevelopmental disorders with poor social interaction, communication issues, aberrant motor movements, and limited repetitive interests and behaviour. Spirulina platensis (SP) contains several multi-nutrients and has a wide range of neuroprotective properties. Aim: The target of the current experiment is to detect the protective effects of S. platensis on valproic-induced autism in adult female albino rats’ siblings for the first time. Materials and Methods: Twelve Pregnant rats were separated into four main groups; Group I (control); Group II (S. platensis); Group III (autistic group); and Group IV (autistic SP-treated group). Fifteen offspring pups from each group were sacrificed, brain was divided for biochemical analysis as superoxide dismutase and malondialdehyde were evaluated spectrophotometrically while interleukin-6, interleukin-12, Bcl-2-associated X protein, B-cell lymphoma-2, Beclin-1, brain-derived neurotrophic factor were assessed by ELISA, other division of brain were used for gene expression of PI3k, Akt and mTOR pathway, last division of brain were stained using (H&E) and Giemsa stains. Tumour necrosis factor alpha (TNF- α ) and Synaptophysin (SYN) markers were used for immunohistochemical staining. Results: Autistic Group (III) showed an increment in levels of MDA, IL-6, IL12 and BAX while showing a decrement in SOD, Bcl-2 and Beclin-1 as well as increased PI3k, Akt and mTOR gene expression. Autistic Group (III) also exhibited hypocellularity and disorganization of hippocampal and prefrontal cortex cells. The autistic SP-treated group (IV) showed improvement in these biochemical markers and pathological changes. Our findings suggest that Spirulina platensis will be significant in managing autism.

Titanium vs PEO Surface-Modified Magnesium Plate Fixation in a Mandible Bone Healing Model in Sheep.

Titanium plates are the current gold standard for fracture fixation of the mandible. Magnesium alloys such as WE43 are suitable biodegradable alternatives due to their high biocompatibility and elasticity modulus close to those of cortical bone. By surface modification, the reagibility of magnesium and thus hydrogen gas accumulation per time are further reduced, bringing plate fixation with magnesium closer to clinical application. This study aimed to compare bone healing in a monocortical mandibular fracture model in sheep with a human-standard size, magnesium-based, plasma electrolytic-oxidation (PEO) surface modified miniplate fixation system following 4 and 12 weeks. Bone healing was analyzed using micro-computed tomography and histological analysis with Movat's pentachrome and Giemsa staining. For evaluation of the tissue's osteogenic activity, polychrome fluorescent labeling was performed, and vascularization was analyzed using immunohistochemical staining for alpha-smooth muscle actin. Bone density and bone mineralization did not differ significantly between titanium and magnesium (BV/TV: T1: 8.74 ± 2.30%, M1: 6.83 ± 2.89%, p = 0.589 and T2: 71.99 ± 3.13%, M2: 68.58 ± 3.74%, p = 0.394; MinB: T1: 26.16 ± 9.21%, M1: 22.15 ± 7.99%, p = 0.818 and T2: 77.56 ± 3.61%, M2: 79.06 ± 4.46%, p = 0.699). After 12 weeks, minor differences were observed regarding bone microstructure, osteogenic activity, and vascularization. There was significance with regard to bone microstructure (TrTh: T2: 0.08 ± 0.01 mm, M2: 0.06 ± 0.01 mm; p = 0.041). Nevertheless, these differences did not interfere with bone healing. In this study, adequate bone healing was observed in both groups. Only after 12 weeks were some differences detected with larger trabecular spacing and more vessel density in magnesium vs titanium plates. However, a longer observational time with full resorption of the implants should be targeted in future investigations.

The involvement of krüppel-like transcription factor 2 in megakaryocytic differentiation induction by phorbol 12-myrestrat 13-acetate

BackgroundMegakaryocytic differentiation is a complicated process regulated by a series of transcription factors in a context- and stage-dependent manner. Recent studies have suggested that krüppel-like transcription factor 2 (KLF2) is involved in the control of embryonic erythroid precursor cell differentiation and maturation. However, the function and mechanism of KLF2 in regulating megakaryocytic differentiation remain unclear.MethodsThe expression patterns of krüppel-like transcription factors (KLFs) during megakaryocytic differentiation were identified from public databases. Phorbol 12-myristate 13-acetate (PMA) treatment of the myeloid-erythroid-leukemic cell lines K562 and HEL were used as cellular megakaryocytic differentiation models. A lentiviral transduction system was utilized to achieve the goal of amplifying or reducing KLF2. The expression of KLF2 was examined using real-time PCR and western blot. The impact of KLF2 on the megakaryocytic differentiation of K562 cells was examined by flow cytometry, Giemsa staining, Phalloidin staining and western blot. RNA-sequencing (RNA-seq) and chromatin immunoprecipitation-sequencing (ChIP-seq) technologies were used to identify the KLF2-regulated targets.ResultsKLF2 is increased in the maturation process of megakaryocytes. KLF2 overexpression accelerated the PMA-induced megakaryocytic differentiation, as reflected by an increased percentage of CD41/CD61 cells, an increased number of polyploid cells, and an elevated expression of P21 and P27. KLF2 knockdown exhibited the opposite results, indicating that KLF2 knockdown suppressed the megakaryocytic differentiation. Further, combination of the RNA-seq and ChIP-seq results suggested that chimerin 1 (CHN1) and potassium voltage-gated channel subfamily Q member 5 (KCNQ5) may be target genes regulated of KLF2. Both CHN1 and KCNQ5 knockdown could block the megakaryocytic differentiation to some content.ConclusionThis study implicated a regulatory role of KLF2 in megakaryocytic differentiation, which may suggest KLF2 as a target for illness with abnormal megakaryocytic differentiation.

The therapeutic efficacy of neem (Azadirecta indica) leaf extract against coinfection with Chlamydophila psittaci and low pathogenic avian influenza virus H9N2 in broiler chickens

Avian chlamydiosis is a serious avian infection that carries a significant zoonotic danger to the poultry industry. The respiratory co-infections caused by the low pathogenic avian influenza virus H9N2 (LPAIV H9N2) also cause significant financial losses in the poultry industry. The purpose of this study was to examine the pathogenicity of Chlamydophila psittaci, and LPAIV H9N2 individually and in combination in broiler chickens, as well as to determine whether or not aqueous neem (Azadirachta indica) leaf extract is effective against infections caused by these pathogens. Therefore, 120 broiler cobb chicks were equally divided into 4 groups (30 birds each) with triplicates with 10 birds. Broilers in group 1 (G1) were infected with only C. psittaci, broilers in group 2 (G2) were infected with only LPAIV H9N2, broilers in group 3 (G3) were infected with C. psittaci and LPAIV H9N2, and broilers in group 4 (G4) remained not challenged and non-treated with any therapeutic or preventive treatment (negative control). At 21 d postinfection (dpi), birds in G1, G2, and G3 were divided into 3 subgroups of 10 birds each: subgroup (A) remained infected and untreated (positive control), subgroup (B) infected and received oxytetracycline for 5 consecutive d, and subgroup (C) infected and received 8% aqueous neem leaf extract for 5 consecutive d. The multiplication of C. psittaci in birds in G1, in various tissues was evaluated using Giemsa staining and the data showed that multiplication was much higher in the lung, spleen, and liver from 6 h to 21 dpi, but low in the heart from 8 to 21 dpi. During simultaneous co-infection in G3, the birds developed significant clinical symptoms and postmortem lesions (PM). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect viral shedding from oropharyngeal and cloacal swabs between 2 dpi and 8 dpi, with cycle threshold (CT) values ranging from 22 to 24. In contrast, bacterial shedding began 6 h after infection and continued until 21 dpi, with CT values ranging from 23 to 26. Administration of an aqueous neem leaf extract at an 8% concentration (Group C) resulted in a numerical rise in average body weight across all treatment groups in the third and fourth week, as well as a reduction in LPAIV H9N2 and C. psittaci replication in the respiratory and gut of treated birds compared to those treated with oxytetracycline (Group B). Overall, respiratory co-infections pose a considerable risk to the poultry business, which is a big threat. To control C. psittaci and LPAIV H9N2 in broiler chickens, oral supplementation of 8% aqueous neem leaf extract is recommended. This treatment improves the birds' performance, as evidenced by an increase in their average body weight. In addition, the application of 8% aqueous neem leaf extract lowers C. psittaci replication within tissues and diminishes LPAIV H9N2 shedding.

Cytogenetic validation of DS02R1-estimated dose for atomic bomb survivors in Hiroshima and Nagasaki with FISH

Introduction For Hiroshima and Nagasaki survivors, it has not been possible to calculate individual doses from the cytogenetic data and compare them with the physically estimated doses. This is because the cytogenetic studies used solid Giemsa staining which only provides the percent of cells bearing at least one stable-type aberration (most of the unstable-type aberrations had already disappeared), and a gamma-ray dose plus a 10-times neutron dose was used to integrate the data for both cities. Objectives To compare the FISH-derived gamma-ray dose with the DS02R1-derived gamma-ray dose after correcting for a contribution of the neutron dose. It was also an attempt to determine if the frequency of stable-type aberrations had remained unchanged after the exposure. Methods Stable exchange-type aberration data was obtained using the 2-color FISH method from 1,868 atomic bomb survivors in Hiroshima and Nagasaki. The collected frequency was first extended to a genome-equivalent frequency. Then, by using known induction rates of exchange-type aberrations in vitro caused by neutrons and gamma-rays, respectively, and the mean relationship between the neutron and gamma-ray doses in the DS02R1 estimates for the survivors, the gamma-ray effect was estimated from the total yield of translocations. Results It was found that over 95% of individual cytogenetic gamma-ray doses fell within the expected range of plus/minus about 1 Gy from the DS02R1 dose and the mean slope for the linear regression was 0.98, which reassures us of the validity of the DS02R1 study. Conclusions The present results demonstrate the validity of the individual DS02R1 doses, and that the frequency of stable-type aberrations in blood lymphocytes did not decay over the years, and thus is useful for retrospective dose evaluations of exposures which took place in the distant past.

Optimization of spermatozoa analysis in mice: A comprehensive protocol

Sperm quality is critical to predict reproductive alterations caused by immunological factors or toxicant agents. Yet, no detailed protocol has been published focusing on analyses of sperm parameters in mice. Our aim was to evaluate the most efficient diluent for mice sperm analyses and to optimize the sperm morphology classification, through the comparison of different staining methods. The diluents assessed were PBS (baseline), HTF, DMEM, 1 % BSA in PBS and 9 % skimmed powdered milk diluted in PBS. Spermatozoa were evaluated for vitality, motility, and morphology, smears were stained with Papanicolaou, HE, Giemsa, and Rapid staining. Sperm vitality and total motility reached better scores in milk based and DMEM diluents. HE raised up as an effective option since its combination with any of the diluents we tested, resulted in a fair staining, which was appropriated to evaluate mice spermatozoa. Finally, based on WHO manual, we have updated the current morphological classification for mice sperm, since we have detailed the head defects as well as included midpiece and tail defects on it. Taken together, we presented a useful, low cost, and reliable method to assess sperm morphology that could be employed worldwide by laboratories dedicated to study reproductive biology on mice model.

Therapeutic potential of Garcinia kola against experimental toxoplasmosis in rats.

Cerebral toxoplasmosis, the most common opportunistic infection in immunocompromised individuals, is increasingly reported in immunocompetent individuals due to mutant strains of Toxoplasma gondii, which, furthermore, are reported to be resistant to available treatments. We assessed the therapeutic potential of Garcinia kola, a medicinal plant reported to have antiplasmodial and neuroprotective properties, against experimental toxoplasmosis in rats. Severe toxoplasmosis was induced in male Wistar rats (156.7 ± 4.1 g) by injecting them with 10 million tachyzoites in suspension in 500 µl of saline (intraperitoneal), and exclusive feeding with a low-protein diet [7% protein (weight by weight)]. Then, animals were treated with hexane, dichloromethane, and ethyl acetate fractions of Garcinia kola. Footprints were analysed and open-field and elevated plus maze ethological tests were performed when symptoms of severe disease were observed in the infected controls. After sacrifice, blood samples were processed for Giemsa staining, organs were processed for haematoxylin and eosin staining, and brains were processed for Nissl staining and cell counting. Compared with non-infected animals, the infected control animals had significantly lower body weights (30.27%↓, P = 0.001), higher body temperatures (P = 0.033) during the sacrifice, together with signs of cognitive impairment and neurologic deficits such as lower open-field arena centre entries (P < 0.001), elevated plus maze open-arm time (P = 0.029) and decreased stride lengths and step widths (P < 0.001), as well as neuronal loss in various brain areas. The ethyl acetate fraction of Garcinia kola prevented or mitigated most of these signs. Our data suggest that the ethyl acetate fraction of Garcinia kola has therapeutic potential against cerebral toxoplasmosis.

Spermocytogram: Comparison Between the Papanicolaou Staining and Modified May Grünwald Giemsa Staining

Introduction: The research and distribution of sperm abnormalities by the spermocytogram are an essential examination in the diagnosis of male infertility. The standard Papanicolaou staining method, recommended by the World Health Organization, for performing a spermocytogram, is too expensive in Congo. The work aimed to look for abnormalities in sperm forms by two comparative methods: Papanicolaou staining and modified May Grünwald Giemsa staining (less expensive), set up in our laboratories.Materials and methods: A series of one hundred spermocytograms were performed by two staining methods: the Papanicolaou method and the modified May Grünwald Giemsa method. The results of two techniques were compared qualitatively and quantitatively.Results: The mean detection of sperm form abnormalities was higher by the Papanicolaou method. However, the precision of the kind of abnormalities highlighted by the two techniques was superimposable.Conclusion: Despite the superiority of the Papanicolaou staining method, which is a reference technique of the spermocytogram, the modified May Grünwald Giemsa staining method can be a first-line technique in the male diagnosis of infertility, especially in the deep Congo. It is efficient, reliable, inexpensive and easily achievable.

The Relative Frequency of Histomonas meleagridis Infection in Turkey Flocks in Some Provinces of Iran

Background: Histomoniasis is caused by the protozoan Histomonas meleagridis with an intermediate host of Heterakis gallinarum, which results in ulceration of the ceca walls, enlargement of the ceca by large casts, mesenteric inflammation, and liver necrosis. This disease is very important in Iran’s growing turkey breeding industry. Objectives: The present study aims to evaluate the relative frequency of H. meleagridis infection in different turkey flocks to draw a cross-sectional picture of H. meleagridis infection in Golestan, Mazandaran, Guilan, and Tehran provinces of Iran. Methods: This study is a cross-sectional survey of H. meleagridis infection during spring. Dropping samples were taken from backyard and commercial turkey flocks. After taking the fecal samples, they were investigated by Giemsa staining under a light microscope. A PCR test was performed to confirm the diagnosis of infection. Results: Out of 240 samples (from 19 flocks), 20 infected samples were detected by direct microscopic observation of H. meleagridis, and PCR confirmed 15 samples. Conclusion: The results of this study showed that the relative frequency of H. meleagridis infection was lower than in similar studies in other parts of the world. This finding may be due to Iran’s less widespread use of turkey production. Considering the growth of the turkey production industry in Iran over the last decade and its further production over the next few years, it is necessary to evaluate histomoniasis.

Plasmodium gametocyte carriage in humans and sporozoite rate in anopheline mosquitoes in Gondar zuria district, Northwest Ethiopia.

Although the overall burden of malaria is decreasing in Ethiopia, a recent report of an unpredictable increased incidence may be related to the presence of community-wide gametocyte-carrier individuals and a high proportion of infected vectors. This study aimed to reveal the current prevalence of gametocyte-carriage and the sporozoite infectivity rate of Anopheles vectors for Plasmodium parasites. A community-based cross-sectional study was conducted from May 01 to June 30/2019. A total of 53 households were selected using systematic random sampling and a 242 study participants were recruited. Additionally,515 adult female Anopheles mosquitoes were collected using Center for Diseases Control and Prevention (CDC) light traps and mouth aspirators. Parasite gametocytemia was determined using giemsa stain microscopy, while sporozoite infection was determined by giemsa staining microscopy and enzyme linked immunosorbent assay (ELISA). Among the total 242 study participants, 5.4% (95%, CI = 2.9-8.3) of them were positive for any of the Plasmodium species gametocyte. Furthermore, being female [AOR = 15.5(95%, CI = 1.71-140.39)], age group between 15-29 years old [AOR = 16.914 (95%, CI = 1.781-160.63)], no ITNs utilization [AOR = 16.7(95%, CI = 1.902 -146.727)], and high asexual parasite density [(95%, CI = 0.057-0.176, P = 0.001, F = 18.402)] were identified as statistically significant factors for gametocyte carriage. Whereas sporozoite infection rate was 11.6% (95%, CI = 8.2-15.5) and 12.7% (95%, CI = 9.6-16.3) by microscopy and ELISA, respectively. Overall, this study indicated that malaria remains to be an important public health problem in Gondar Zuria district where high gametocyte carriage rate and sporozoite infection rate could sustain its transmission and burden. Therefore, in Ethiopia, where malaria elimination program is underway, frequent, and active community-based surveillance of gametocytemia and sporozoite infection rate is important.

The Trypanosoma cruzi pleiotropic protein P21 orchestrates the intracellular retention and in-vivo parasitism control of virulent Y strain parasites.

P21 is a protein secreted by all forms of Trypanosoma cruzi (T. cruzi) with recognized biological activities determined in studies using the recombinant form of the protein. In our recent study, we found that the ablation of P21 gene decreased Y strain axenic epimastigotes multiplication and increased intracellular replication of amastigotes in HeLa cells infected with metacyclic trypomastigotes. In the present study, we investigated the effect of P21 in vitro using C2C12 cell lines infected with tissue culture-derived trypomastigotes (TCT) of wild-type and P21 knockout (TcP21-/-) Y strain, and in vivo using an experimental model of T. cruzi infection in BALB/c mice. Our in-vitro results showed a significant decrease in the host cell invasion rate by TcP21-/- parasites as measured by Giemsa staining and cell count in bright light microscope. Quantitative polymerase chain reaction (qPCR) analysis showed that TcP21-/- parasites multiplied intracellularly to a higher extent than the scrambled parasites at 72h post-infection. In addition, we observed a higher egress of TcP21-/- trypomastigotes from C2C12 cells at 144h and 168h post-infection. Mice infected with Y strain TcP21-/- trypomastigotes displayed higher systemic parasitemia, heart tissue parasite burden, and several histopathological alterations in heart tissues compared to control animals infected with scrambled parasites. Therewith, we propose that P21 is important in the host-pathogen interaction during invasion, cell multiplication, and egress, and may be part of the mechanism that controls parasitism and promotes chronic infection without patent systemic parasitemia.

Retrospective analysis of laboratory diagnostic data to assess the prevalence and seasonal variation of Ehrlichia ruminantium in commercial farms of Lephalale Municipality, Limpopo, South Africa

Heartwater is an economically important disease of livestock and some wild ruminants in Southern Africa. The study used retrospective laboratory data from Lephalale Veterinary Laboratory, Limpopo to establish the prevalence and seasonal occurrence of heartwater in commercial farms of Lephalale Municipality between 2010 and 2022. A total of 472 brain samples from livestock (cattle, goats, sheep) and game carcasses brought to the laboratory for postmortem examination were subjected to heartwater testing using Giemsa staining technique. The overall prevalence of heartwater in Lephalale Municipality was 34.1% (95% CI = 29.9–38.6, p < 0.001). During the years under investigation, there was a significant (p < 0.001) variation, with the highest and lowest prevalence in 2022 (72.2%) and 2019 (6.3%), respectively. The prevalence of heartwater was high in springbok (57.1%), sheep (49.3%), cattle (36.6%) and goats (32.2%). Heartwater was detected throughout the year with autumn having the highest (40.6%) frequency followed by summer (40.3%), spring (30.5%) and winter (25.5%). This study is the first to establish the prevalence and seasonality of heartwater in Lephalale Municipality which will serve as a baseline for prevention and control strategies as well as future epidemiological studies. Official surveillance programmes, more research on the distribution and genotypes of E. ruminantium in the area need to be undertaken for better understanding of the disease in the area.

Efficacy of a Combined Herbal Product Containing Indigofera argentea, Pistacia atlantica and Salvia hispanica in Zoonotic Cutaneous Leishmaniasis Patients Treated along Glucantime ®.

Leishmaniasis is highly prevalent worldwide, and while conventional medicine offers numerous treatment methods for cutaneous leishmaniasis, Iranian traditional medicine suggests various remedies. We aimed to evaluate the efficacy of an herbal combination containing Indigofera argentea leaves, Pistacia atlantica resin, and Salvia hispanica seeds in patients with zoonotic cutaneous leishmaniasis. This study was conducted at the Leishmaniasis Department of Chabahar Health Center in southeastern Iran in 2021. It was a double-blinded randomized clinical trial involving 68 patients enrolled after clinical diagnosis, examination of smear samples using Giemsa staining, and PCR confirmation. The volunteers were randomly divided into intervention and control groups. Both groups received 'Glucantime ® as the primary medication weekly until complete healing or up to 12 weeks. Glucantime ® was administered intralesionally at a dosage of 0.1 cc on the wound's margin, with repeat injections at 1 cm intervals along the wound edge when necessary. In addition to the main drug, the intervention group received the herbal product topically twice daily until wound healing or for up to 4 weeks, while the control group received a placebo in the same manner. 82.35% of patients in the intervention group and 20.58% in the control group achieved complete healing within four weeks. There was a significant difference between the two groups (P < 0.001). The herbal product demonstrated effectiveness in treating patients with zoonotic cutaneous leishmaniasis.

Validation of a novel alcohol-free preservative solution in comparison with conventional prefixation on quality of serous body fluid smears.

We describe a novel alcohol-free preservative composed of glucose, mannitol, disodium hydrogen orthophosphate, thymol, and distilled water (glucose-mannitol-disodium dihydrogen orhtophosphate-thymol [GMDT] preservative) in appropriate proportion as an alternative to alcohol prefixation (APF) of body fluids. To assess the cytomorphologic preservation and staining quality of serous body fluid smears generated by GMDT preservative and compare it with smears processed by standard 50% APF. The study comprised 151 effusion samples. Each sample was equally divided into four tubes. Equal volumes of APF and GMDT preservatives were added to the first two tubes and left at room temperature for 24 h. Similarly, the corresponding preservatives were added to the third and fourth tubes and stored for 48 h. Two smears were prepared from the centrifuged sediments of each tube (all four tubes) and stained with May-Grünwald Giemsa and Papanicolaou (Pap) stains. Using a three-tiered scoring system, the smear examination was blinded to assess the extent of cellular preservation and the staining quality by two cytotechnologists and two cytopathologists. Statistical analysis was performed by STATA 16.0. Samples processed with the GMDT preservative at 24 h showed better cytoplasmic preservation and smear background, while nuclear features and staining quality showed no difference between the two preservatives. Mild cytoplasmic and nuclear degenerative changes were noted with the GMDT at 48 h, while all four parameters remained similar with APF at 24 and 48 h. The newly developed alcohol-free, GMDT preservative, could be a feasible and cost-effective alternative to 50% APF, preferably when samples are processed within 24 h.

Accumulation and Phagocytosis of Fluorescently Visualized Macrophages Against Edwardsiella piscicida Infection in Established mpeg1.1-Transgenic Japanese Medaka Oryzias latipes.

Intracellular bacteria such as those belonging to the genus Edwardsiella can survive and proliferate within macrophages. However, the detailed mechanisms underlying the host macrophage immune response and pathogen evasion strategies remain unknown. To advance the field of host macrophage research, we successfully established transgenic (Tg) Japanese medaka Oryzias latipes that possessesfluorescently visualized macrophages. As a macrophage marker, the macrophage-expressed gene 1.1 (mpeg1.1) was selected because of its predominant expression across various tissues in medaka. To validate the macrophage characteristics of the fluorescently labeled cells, May-Grünwald Giemsa staining and peroxidase staining were conducted. The labeled cells exhibited morphological features consistent with those of monocyte/macrophage-like cells and tested negative for peroxidase activity. Through co-localization studies, the fluorescently labeled cells co-localized with E. piscicida in the intestines and kidneys of infected medaka larvae, confirming the ingestion of bacteria through phagocytosis. In addition, the labeled cells expressed macrophage markers but lacked a neutrophil marker. These results suggested that the fluorescently labeled cells of Tg[mpeg1.1:mCherry/mAG] medaka were monocytes/macrophages, which will be useful for future studies aimed at understanding the mechanisms of macrophage-mediated bacterial infections.

(032) A REVIEW OF VESTIBULECTOMY PATHOLOGY FINDINGS AND A CALL TO ACTION TO STANDARDIZE PATHOLOGY REPORTING

Abstract Introduction There is no standardization for pathology reporting of vestibulectomy specimens. Our current understanding of neuroproliferative vestibulodynia is limited, but it is understood to be mediated by the accumulation c-afferent nociceptors and mast cells. Histopathology is fundamental to our understanding of disease mechanism and treatment. Given how much remains unknown about the histology, physiology, and neuroplasticity of the vestibule, it is imperative that we take every opportunity to fully examine this tissue and its complex role in pain. Objective Describe current pathology reporting practices for vestibulectomy specimens Methods Data was collected using Epic and Cerner report tools to identify any patients between 1/2017 to 10/2023 with a surgical history of vestibulectomy. Forty-five patients were identified &amp; confirmed to have a history of vestibulectomy. Charts were reviewed to locate surgical pathology reports (available through hospital EMR, CRISP, or Care Everywhere). Results Pathology reports were available for 37 patients. Thirty-one of these reports (84%) were from our own institution; six reports were from outside hospitals. Marked variability in pathology reporting was noted between pathologists and institutions. All but one pathology report notes inflammation. The one report that does not include language about inflammation only notes “negative for dysplasia or malignancy”. Twenty-nine reports comment on the absence of dysplasia, including two reports from other institutions. All pathology reports from our institution (100%), note lymphoid infiltrate; however, this is not noted on any of the 6 pathology reports from other hospitals. Nineteen of the 31 specimens (61%) examined at our institution included specific stains to test for mast cells and nerve fibers. Nineteen specimens were evaluated with Giemsa staining and 8 specimens were evaluated with CD117 staining to assess for mast cell density (measured in mast cells/hpf). The range of mast cell density was 1/hpf to 25/hpf, and the average mast cell density was 8.1/hpf. All 19 specimens stained with S100 noted a relative increase in nerve fibers in the superficial dermis. Conclusions Histopathology is a window of opportunity to better understand the pathophysiology of neuroproliferative vestibulodynia. There is no standard when it comes to examining and reporting on vestibulectomy specimens. This results in reporting inconsistencies, which limits our ability to compile data and draw clinical conclusions. As surgeons we can collaborate to advance our understanding of the pathophysiology of vestibulodynia. This starts with communicating with our pathology colleagues, requesting appropriate stains, and setting a standard for reporting. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Patty Brisben Foundation.

Effect of electroacupuncture on autophagy of ovarian granulosa cells in rats with premature ovarian insufficiency

To observe the effects of electroacupuncture (EA) on the autophagy of ovarian granulosa cells in rats with premature ovarian insufficiency (POI), and explore the mechanism of EA in improving POI. Thirty-two female SD rats were randomly divided into a blank group (n=8) and a model making group (n=24). The rats in the model making group were injected intraperitoneally with cyclophosphamide for 15 days to establish the POI model (the dosage on the 1st day was 50 mg/kg, and 8 mg/kg from the 2nd day to 15th day). The successfully modeled rats were then randomly divided into a model group, an EA group, and an estradiol (E2) group, with 8 rats in each group. Rats in the EA group received EA at bilateral "Gongsun" (SP 4) with continuous wave, frequency of 2 Hz, and current intensity of 0.1 to 1 mA, 20 min per treatment, once daily for 14 days. Rats in the E2 group were administered with E2 (0.01 mg/mL) by gavage (10 mL/kg), once daily for 14 days. The changes in estrous cycle were observed by rapid Giemsa staining before and after modeling. After intervention, ovarian tissue morphology was observed by HE staining; serum levels of follicle-stimulating hormone (FSH), E2, anti-Mullerian hormone (AMH), and inhibin B (INHB) were detected by ELISA; immunofluorescence staining was used to observe the expression of p62 in ovarian granulosa cells; the ultrastructure of ovarian granulosa cells was observed by transmission electron microscopy, and the number of autophagosomes and autolysosomes was compared; Western blot and real-time fluorescence quantitative PCR were used to detect the protein and mRNA expression of p62, Beclin-1, and microtubule-associated protein 1A/1B-light chain 3 (LC3) in ovarian tissue. The results of vaginal smears in the blank group showed regular cyclical changes; the rats in the model group showed prolonged estrous cycle or cycle arrest, mostly in proestrus or metestrus, with overall ovarian atrophy, disordered structure, and decreased granulosa cells. Compared with the blank group, rats in the model group showed increased serum FSH level (P<0.01), decreased serum levels of E2, AMH, and INHB (P<0.01), decreased positive expression of p62 in ovarian granulosa cells (P<0.01), with obvious swelling of ovarian granulosa cells, mild to moderate swelling of mitochondria, slight expansion of rough endoplasmic reticulum, and hypertrophy of Golgi apparatus; the number of autophagosomes and autolysosomes in the ovaries was increased (P<0.01), the expression of p62 protein and mRNA was decreased (P<0.01), and the expression of Beclin-1 and LC3 protein and mRNA in ovarian tissue was increased (P<0.01). Compared with the model group, rats in the EA group and the E2 group showed decreased serum FSH levels (P<0.01), increased levels of E2, AMH, and INHB (P<0.01), increased positive expression of p62 in ovarian granulosa cells (P<0.01), alleviated degree of ovarian granulosa cell damage, with relatively intact organelle morphology, and decreased number of autophagosomes and autolysosomes in the ovaries (P<0.01); the rats also showed increased expression of p62 protein and mRNA (P<0.01), and decreased expression of Beclin-1 and LC3 protein and mRNA (P<0.01) in ovarian tissue. EA at "Gongsun" (SP 4) could improve ovarian reserve function in POI rats by reducing the number of autophagosomes and autolysosomes, up-regulating p62 expression, and down-regulating Beclin-1 and LC3 expression, thus inhibiting autophagy of ovarian granulosa cells, and regulating the serum levels of FSH, E2, AMH, and INHB.