CATSPER 1 (Cation Channel Sperm Associated 1) and CATSPER2 channels have an important role in sperm motility. In this study, the effects of hyperthyroidism on Catsper1 and 2 genes of seminiferous tubules in mice testes were investigated.CATSPER1 and CATSPER2 genes in the seminiferous tubules of mice.The present study was conducted to investigate the effect of hyperthyroidism on the expression of This study was conducted on 20 BALB/C male mice divided into two groups - experimental and control. The experimental group was administered with 500 mg/l levothyroxine (L-thyroxine) liquid solution for two months for inducing hyperthyroidism, which was confirmed by radioimmunoassay. On the other hand, the control group was kept in animal houses under a normal condition. The implementation of real-time polymerase chain reaction and immunohistochemical studies was accomplished after the removal of the testes of the mice under anesthesia induced by chloroform.CATSPER1 (p = 0.45) and CATSPER2 (p = 0.34) gene expression between groups. At the same time, the color intensity showed no significant enhancement in the hyperthyroidism group (CATSPER1 p = 0.17 and CATSPER2 p = 0.22) as compared to the control group.Results showed that there was no significant difference in CATSPER in the molecular structure of the sperm, our findings showed that the hyperactivity of the thyroid gland has no significant effects on the function of these components. Therefore, it might be concluded that hyperthyroidism has no considerable effects on the seminiferous tubules.Considering the key role of  The endocrine system is the second most important regulatory system in the body after the nervous system. Thyroid-stimulating hormone (THS) stimulates the thyroid gland to secrete thyroxin, and then triiodothyronine hormones. Although THS plays different roles in many cells and tissues of the body, the function of this hormone in sperm development requires further research (1). Thyroid hormone activates the function of a wide range of genes that guide nuclear receptors through complicated signaling pathways (2). The testicles are mainly composed of seminiferous tubules supported by the interstitial connective tissue. Each tubule consists of three layers, namely basement membrane, myoid cells, and elastic fibers. Sertoli cells in BM play multiple roles, including the establishment of a blood-testis barrier (3). Excessive thyroid hormones cause hyperthyroidism, which is associated with decreased testicular mass, testosterone level, and Sertoli cells . Thyroid hormone receptors (TR) are located on Sertoli cells. Different patterns of TR gene expression include TRα and TRβ, which are the main factors in the regulation of the effect of the thyroid hormones on the testes (6-8).CATSPER) is a type of voltage-sensitive ion channel that allows the entrance of calcium ions. Sperm motility is the most important factor in fertilization, which is mainly regulated by calcium signaling . CATSPER1 is found in the tail of the sperm, and CATSPER2 is required for the entrance of calcium ions to the sperm (13-15). The CATSPER gene encodes a six-transmembrane-spanning repeat that its product are only expressed in testes. CATSPER gene produces the four repeat structure of voltage-dependent CaCATSPER gene reduced sperm motility and male fertility function for sperm penetration of the zona pellucida in mice. In addition, investigation showed that hyperactivated motility needs a sperm-specific pH-gated calcium channel in mice. So that increasing intracellular pH induced a rise in intracellular calcium. The amount of calcium regulate sperm motility through the motor proteins of the tail sperm .Mammalian fertilization is one of the most amazing biological phenomena, involving the infusion of the sperm with the oocyte. Calcium channels play an important role in various mammalian sperm functions, such as capacitation and acrosome reaction . The cation channel of sperm (CATSPER1 and CATSPER2) in motility and the function of sperm, the aim of the present study was to investigate the effect of hyperthyroidism on the expressions of CATSPER1 and CATSPER2 genes in the seminiferous tubules of mice.According to the importance of cation channel . During the experiment, the mice had enough access to food and water. All protocol of immunohistochemistry and real-time PCR were carried out at a specialized laboratory of histochemistry and Buali (Avicenna) at the Research Institute of Mashhad University of Medical Sciences.After the adaptation to the environment, the mice were randomly divided into two groups of hyperthyroidism and control. The control did not receive any interventions, while the hyperthyroidism group was administered with 500 mg/l levothyroxine (L-thyroxine) liquid solution for two 2 months (18). After the treatment period, the hyperthyroidism was confirmed by means of radioimmunoassay after three days in laboratory. Then the mice were kept in the normal conditions of the animal house. After four days, all mice were sacrificed, and their testes were extracted and fixed in formalin. Based on the common histological methods, the samples were molded in paraffin.CATSPER1 and CATSPER2 immunohistochemical investigation.For supplying smear, epididymis were removed and divided into small pieces and incubated in 1 ml normal saline, 5% COCATSPER1 or CATSPER2 antibodies were deposited on the samples at a concentration of 1:50 and then incubated (overnight) at 40° for 24 hr. After washing the samples with PBS for 15 min, they were incubated with the donkey anti-goat polyclonal secondary antibody at a concentration of 1: 200 at 37°C for 2 hr. The samples were washed again with PBS, and then placed in a solution containing 0.3 g of 3,3'-diaminobenzidine, 200 μl water, oxygenated water, and PBS for 15 min. After washing the specimens with running water for 1.5 min, they were rinsed with water for 12 min. The samples were then digested using ascending alcohol series (700-1000). Finally, they were sealed for clarification in xylene and between the slide and coverslip. To study the brown color intensity of the samples, an optical microscope  was connected to a camera with a 100CATSPER1 or anti-CATSPER2 considering light to dark brown staining based on their immunohistochemically staining intensity.The animals were sacrificed through cervical dislocation under anesthesia induced with chloroform; subsequently, the epididymis was removed, followed by the incubation of the epididymis fragments mixed with physiological serum at 370°C for 30 min. Next, 10 μl of the suspension containing sperm was spread on a poly-L-lysine slide and then fixed with methanol. For immunohistochemical study, the smears were washed with phosphate-buffered saline (PBS) thrice for 15 min. Subsequently, a standard method was applied for performing heat-induced antigen retrieval. After placing the samples in EDTA/PBS solution at 100°C for 30 min, they were washed in PBS for 15 min. In order to control the peroxidase activity, the samples were immersed in 3% oxygen water, in addition to methanol in darkness for 20 min. Then, they were incubated with PBS for 15 min and 1.5% bovine serum albumin buffer for 30 min at room temperature. Next, the primary CATSPER1 and CATSPER2 were examined by the SYBR Green quantitative real-time polymerase chain reaction kit  using the mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as the housekeeping gene.Total RNA was isolated from the testicular samples by means of the Total RNA Purification kit  following the manufacturer's instructions. The integrity and purity of the obtained RNA were evaluated using electrophoresis on 1.5% agarose gel  and visualized by an ultraviolet transilluminator. The synthesis of the first-strand complementary DNA (cDNA) of the extracted messenger RNA (mRNA) was performed using the Easy cDNA Synthesis kit in the presence of Oligo d (T)CATSPER1 and CATSPER2 genes were normalized to the Ct of the housekeeping gene . The relative quantification value of the target is expressed as follows:Table II lists the primers applied in the present research. Each reaction (10 µl) contained 5 µl SYBR 2X Mater Mix and 0.5 µM of each specific primer. Amplification was performed with an initial denaturation at 95°C for 10 min, followed by 45 cycles of amplification at 94°C for 20 sec and 60°C for 30 sec using the Rotor-Gene Q . The reaction mixtures were warmed up at 72°C for 3 min; subsequently, the temperature was ramped to 99°C to analyze the melting curves. To evaluate the gene expression, the cycle thresholds (Ct) for 2ΔCt = CT (target sample) - CT (Referenses)ΔΔCt = ΔCT (target sample) - ΔCT (control sample)Ratio= 2The animals were used in accordance with the standards of the Animal Research Ethics Committee and accepted by the Ethics Committees of Mashhad University of Medical Sciences (No: IR.MUMS.fm.REC.1394.306).t test was used for gene expression in RT-PCR. investigations. The data on average Standard deviation . According to this software, The Mann-Whitney test was used for immunohistochemically reactions and CATSPER1, there was no reaction in all the three segments of the spermatozoa in the hyperthyroidism group. However, this intensity of coloration was clearly observed in all the three parts of spermatozoa in the control group. In this regard, the head and middle pieces showed high reactions, and the tail demonstrated a moderate reaction . Considering CATSPER2 in the hyperthyroidism group, the severity of the immunohistochemical staining of the head, middle piece of the sperms was moderate, while that of the tail of the spermatozoa was obtained as low. In the control group, the head and middle piece showed high reactions, while the tail demonstrated a moderate reaction. This comparison showed variation in color from bright to dark brown . The results of the Mann-Whitney U-test revealed no significant differences between the hyperthyroidism and control groups in terms of the severity of immunohistochemical reactions in both CATSPER1 (p = 0.17) and CATSPER2 (p = 0.22). Similarly, the results of the t test also showed no significant difference between the hyperthyroidism and control groups regarding the real-time PCR results of the mRNA gene expression in both CATSPER1  and CATSPER2 .After the confirmation of hyperthyroidism in the experimental mice by the relevant tests, the intensity of immunohistochemical staining was determined in the three parts of the sperm  for both hyperthyroidism and control groups. With regard to CATSPER in the molecular and functional structure of the sperm and its motility (11), the present study was targeted toward the investigation of changes in CATSPER gene and protein in hyperthyroidism cases. In our study, not only the expression rates of both CATSPER1 and CATSPER2 genes were not significantly different between the hyperthyroidism and control groups, but also the immunohistochemically reactions of anti- CATSPER1 and CATSPER2 antibodies between the two groups. Western blot analysis indicates that the expression level of the CATSPER1 gene is directly related to sperm with progressive motility. In addition, the expression of this gene in sperm with normal seminal fluid environmental conditions increased as compared to sperm that do not have these Optima conditions (20).According to the previous study, hyperthyroidism leads to the reduction of some parameters in the Sertoli and Leydig cells in the testes and cellular function in the spermatogenesis process (19). Considering the significant role of CATSPER1 has a direct relationship with the sperm speed and quality and an inverse relationship with aging. Accordingly, the dead sperms have a low percentage of CATSPER1 expression (21). In a couple of studies performed by Mohammedi and colleagues on male mice treated with cadmium and nickel, there was a significant difference between the experimental and control groups regarding the expressions of CATSPER1 and CATSPER2 genes, which is not similar to our results. Aside from the toxic effects of selenium on most cells and tissues, it seems that it also has its undesirable effects on CATSPER1,2 gene and protein expression . In another study on adult male mice treated with L-carnitine, the expression rate of CATSPER1 protein and immunohistochemical reaction of the sperm  increased in the experimental group compared to those in the control group. They also obtained similar results regarding CATSPER2 protein. On the contrary, our results showed a reduction in staining intensity in the hyperthyroidism mice compared to that in the control group (24). Previous findings showed that in hypothyroid mice, hypothyroidism has an effect on the expression CATSPER1,2 gene and protein that results in significant difference between the experimental group and the control group (25). However, the results of this study on the effects of hyperthyroidism on the aforementioned factors did not show any significant differences in the experimental group compared to the control group. Therefore, it can be concluded that the decrease in thyroid hormone levels may affect the CATSPER1,2 gene and protein expression (25). In varicocele disease, sgnificant increase was observed in the CATSPER1,2 gene and protein expression in the experimental group compared to the control group, which did not match with the results of our research in terms of significance. In this regard, study on varicocele was associated with increased testicular temperature. So, increasing temperature may also, in addition to destructive effect on testicular tissue, effect on increasing significant difference on CATSPER1,2 gene and protein expression (26).Meanwhile, the expression of Blood collection from mice was challenging due to their low blood volume; thus, we used gel tubes provided by the laboratory and performed the required evaluations.CATSPER1 and CATSPER2 genes. In addition, no reaction was observed in the histochemical staining intensity of CATSPER1. Regarding the CATSPER2, the staining intensity in hyperthyroid specimens was weak.As the findings of the present study indicated, hyperthyroidism has no significant changes in the expressions of both There is no conflict of interests.


Oncorhynchus keta. At 3 days after a traumatic brain injury (TBI) in juvenile chum salmon, the GS expression was detected in the radial glia corresponding to aNSC in the pallium and subpallium. The maximum density of distribution of GS+ radial glia was found in the dorsal pallial region. Hydrogen sulfide (H2S) is a proneurogenic factor that reduces oxidative stress and excitotoxicity effects, along with the increased GS production in the brain cells of juvenile chum salmon. In the fish brain, H2S producing by cystathionine β-synthase in neurogenic zones may be involved in maintaining the microenvironment that provides optimal conditions for the functioning of neurogenic niches during constitutive neurogenesis. After injury, H2S can determine cell survivability, providing a neuroprotective effect in the area of injury and reducing the process of glutamate excitotoxicity, acting as a signaling molecule involved in changing the neurogenic environment, which leads to the reactivation of neurogenic niches and cell regeneration programs. The results of studies on the control of the expression of regulatory Sonic Hedgehog genes (Shh) and the transcription factors Paired Box2 (Pax2) regulated by them are still insufficient. A comparative analysis of Pax2 expression in the telencephalon of intact chum salmon showed the presence of constitutive patterns of Pax2 expression in neurogenic areas and non-neurogenic parenchymal zones of the pallium and subpallium. After mechanical injury, the patterns of Pax2 expression changed, and the amount of Pax2+ decreased (p < 0.05) in lateral (Dl), medial (Dm) zones of the pallium, and the lateral zone (Vl) of the subpallium compared to the control. We believe that the decrease in the expression of Pax2 may be caused by the inhibitory effect of the Pax6 transcription factor, whose expression in the juvenile salmon brain increases upon injury.The considerable post-traumatic brain recovery in fishes makes them a useful model for studying the mechanisms that provide reparative neurogenesis, which is poorly represented in mammals. After a mechanical injury to the telencephalon in adult fish, lost neurons are actively replaced due to the proliferative activity of neuroepithelial cells and radial glia in the neurogenic periventricular zone. However, it is not enough clear which signaling mechanisms are involved in the activation of adult neural stem cells (aNSC) after the injury (reactive proliferation) and in the production of new neurons (regenerative neurogenesis) from progenitor cells (NPC). In juvenile Pacific salmon, the predominant type of NSCs in the telencephalon are neuroepithelial cells corresponding to embryonic NSCs. Expression of glutamine synthetase (GS), a NSC molecular marker, was detected in the neuroepithelial cells of the pallium and subpallium of juvenile chum salmon,  Danio rerio [Scyliorhinus canicula [Oncorhynchus masou [Active brain recovery after injury observed in fishes provides a useful model for studying the mechanisms responsible for reparative neurogenesis, while mammals exhibit a reduced neurogenesis capacity. It has been reported that in fish brain the generation of functional neurons from precursors (neurogenesis) and activated stem cells, or transdifferentiation of key elements, occurs during regeneration ,2. In coio rerio , the catcanicula , and masus masou . HoweverEublepharis macularius show the presence of proliferating pools of neural stem/progenitor cells in the septomedialis sulcus, a ventricular zone adjacent to the medial cortex. These cells express sex determining region Y-box 2 (SOX2), glial fibrillar acidic protein (GFAP), and vimentin, and have a radial glial morphology [Scyliorhinus canicula, various proliferating populations of radial glia cells were found in the pial zone [The central nervous system (CNS) in fishes and amphibians has the highest capacity for neurogenesis, with the physiological neurogenesis and transdifferentiation of pre-existing elements launched simultaneously after a traumatic injury . The phyrphology . Nerve tial zone . HoweverIt has been shown that in fish, both in case of induced inflammation and in traumatic brain injury, the proliferation of progenitor cells and, as a consequence, the number of new neurons, increases . This suStudies on mammals have shown that acute inflammation interferes with adult neurogenesis and regenerative processes . Thus, i2S being a candidate for the regulation of intercellular interactions performing the common cytoprotective and signal functions [2S is produced by several pathways, in particular by cystathionine β-synthase (CBS) which is a key enzyme in the formation of cysteine from methionine. This gasotransmitter activates intracellular signaling pathways through the sulfhydration of proteins and can react with protein hemes. There are multiple interplays between H2S and other gasotransmitters both on the level of their generation and as targets [Oncorhynchus masou demonstrated sharply increasing the number of CBS+ cells after 3 days, which indicates the involvement of H2S in the post-traumatic response [2S-producing cells in the integrative centers of the brain: telencephalon, optic tectum, and cerebellum [2S has a significant effect on physiological and pathophysiological processes in the CNS, being involved in protective mechanisms induced by traumatic brain injury (TBI) and ischemic reperfusion [Several key substances induce neurogenesis and reparative processes, with Hunctions ,14. It i targets . Previouresponse . Similarrebellum . A notewrebellum . H2S haserfusion . Acute ierfusion ,21. The erfusion .2S in the processes of ischemic brain injury, TBI and the involvement of this gas transmitter in the control of oxidative stress and the increase in reactive oxygen species in the H2S-dependent signaling are being actively studied [2S reactions with many signaling mediators, transcription factors, and channel proteins are known to occur in neurons and glial cells both in vivo and in vitro [2S in regenerative processes, in particular, in adult neurogenesis and TBI, is still limited.Currently, the involvement of H studied ,22,23. Hin vitro ,19. Howe+ reactive astrocytes in it, being involved in the initial stages of the optic nerve axon regeneration [Adult neurogenesis and neuronal regeneration after injury are controlled by the activation of certain molecular pathways, including transcription factors, growth factors, neurotrophins, and cytokines, which are expressed in certain neurogenic niches and, ultimately, at the damaged CNS site. Shh signaling plays an important role in both CNS neurogenesis and regeneration . Transcrneration . In the neration . It has neration .O. masou is largely due to the presence of tissue-specific precursors of the neuroepithelial type [Oncorhynchus keta and in fish at three days after a mechanical injury.Juvenile Pacific salmon is a convenient model for the study of postembryonic neurogenesis, since the intensive neurogenesis in its brain is provided by neuroepithelial progenitors ,16,28. Tial type . The maiOncorhynchus keta, the dorsal (D) and ventral (V) regions, corresponding to the pallial and subpallial regions of zebrafish, were identified [In the telencephalon of the juvenile chum salmon entified . The dorThe patterns of the GS labeling in the telencephalon of the intact animals related to the matrix periventricular zones A. In the+ cells of the neuroepithelial type were located above the layer of immunonegative cells in the PVZ  differences between the groups of intensively, moderately labeled cells and immunonegative cells in the telencephalon of juvenile chum salmon  C. RG aft+ fibers D. In the+ fibers E. In somd in PVZ .+ cells in such areas differed in morphological heterogeneity and intensity of GS labeling , compared with control animals (p < 0.05); the maximum number of fibers was found in DD3; the minimum, in DD1 ; the maximum number of fibers was localized in DM3; the minimum, in DM1 , three subzones, DM1, DM2, and DM3, were also identified A–C. In Dersisted . PVZ conial type . In PVZ,entified B. In somnt layer B. In DM1nt layer . In someobserved B, inset.observed C. A comp, in DM1 G.+ cells generated a monolayer structurally similar to that in DD, and, thus, DL was also divided into three subregions: DL1, DL2, and DL3 ; the maximum number of RG fibers was found in DL1; the minimum, in DL3  and ventral (VL2) C,D. In Vated PVZ C, inset,ot found D, inset.+ groups of neuroepithelial cells, RG fibers, and single moderately labeled cells of parenchymal localization  and ventral (VV2) E,F. RNNslization . In VV1,lization . In VV2,revealed F.+ cells in the fish on day 3 post-injury showed significant differences in DD, VV, VL (p < 0.05), and DM (p < 0.01) from the control (intact) animals , ventral (VV), and lateral zones (VL) A. The legranules .+ granules were also found in single cells of PVZ, extending deep into SVZ , as well as immunonegative cells  between these groups  and the ventral nucleus of the subpallium (VV) (p < 0.05), as well as in VL (p < 0.01)  in pallial and subpallial populations of intensely/moderately labeled cells  A. In theominated B. Howeveominated C. A comped cells D.+ cells in DD, DM, and DL was represented by a multi-row layer of intensely labeled cells reaching a considerable length, interrupted at the border of the pallial zones  and ventral (VV) nuclei . In PZ oncreased F. The vancreased . In genencreased .The expression patterns of the transcription factor (TF) Pax2 in the telencephalon of the intact animals were not directly related to the matrix periventricular zones and formed morphogenetic fields in which TF expression was present in nuclei and cells . In the + cells were moderately labeled . Significant differences between pallial  and subpallial  cell groups (p < 0.05) were revealed. A densitometric study of the optical density of Pax2+ immunolabeling in the pallium and subpallium showed significant differences between the intensely, moderately, poorly labeled cells and the immunonegative cells  in the medial and lateral areas of the pallium  and the lateral area of the subpallium (VL) after injury (+ cells (p < 0.05) in the pallial and subpallial areas that are not adjacent to the injury area.The data of quantitative analysis showed that the number of Pax2r injury G compare in the telencephalon of juvenile intact and injury-exposed fish showed the characteristic significant structural rearrangements and the appearance of RG cells in the pallial and subpallial parts.+ cells of the NE type have also been found in the dorsolateral pallium of zebrafish [+ cells in the pallium of juvenile chum salmon confirmed the heterogeneous pattern of labeled NPCs. In our studies, GS labeling was found in all pallial zones of the telencephalon. The pattern of labeling made it possible to distinguish both rather extensive neurogenic regions containing GS+ cells and single clusters, morphologically corresponding to local neurogenic niches of the constitutive type.In the intact brain, GS labels a heterogeneous population of NPCs localized in PVZ, as well as a limited number of NPCs in parenchyma. In the medial pallial zone (DM) and all subpallial zones, GS labeling was detected in the granules located in PVZ . In the ebrafish . Howeverebrafish . Previouebrafish . An anal+ cells are concentrated in the medial zone than in the dorsal and lateral zones [To characterize the rate of NPC proliferation and the fate of new neurons, the pallial and subpallial neurogenic zones of fishes are currently considered from the comparative aspect of the subgranular (SGZ) and subventricular (SVZ) neurogenic zones of mammals . Thus, ial zones . Accordial zones ,32.+ cells and resting cells (PCNA–) has been revealed [+ cells are phenotyped as radial glia, both in terms of morphological traits and in positivity for radial glia markers such as glial fibrillar acidic protein (GFAP), vimentin, S100B protein, Notch and HER4, glutamine synthetase, specific astrocyte glutamate transporter, and fatty acid binding protein [+ cells are characterized as differentiating neuroblasts as they express the neural protein polysialylated cell adhesion molecule (PSA-NCAM) [+ cells of the NE type exclusively in the constitutive neurogenic niches (CNN) of the intact juvenile chum salmon pallium.In zebrafish pallium, an alternating pattern of dividing PCNArevealed ,33. Abou protein ,33,34,35SA-NCAM) ,37. In tSA-NCAM) ,32. The + cells is more consistent with the localization of CNN. Thus, we can conclude that in the telencephalon of intact chum salmon GS a population of neuronal progenitor cells is labeled with an NE phenotype.In the subpallial area of juvenile chum salmon, the distribution of GSzonula occludens (ZO1), and parvalbumin (PAR) PAR proteins [The subpallial zone of the fish telencephalon is currently considered as an analogue of the subventricular zone (SVZ) of mammals ,33,35,38proteins . The graproteins .+ neuronal precursors with a radial glia phenotype that is absent in intact animals  increased in both groups vs. the control  and in all areas of the subpallium of juvenile chum salmon  with the RG morphology.As a result of injury to the in trout ,18 and cin trout . Due to r injury . Unlike 2S on voltage-gated ATP-sensitive potassium channels KATP and fibrosis transmembrane conductance regulator CFTR Cl-channels [2S in glutamate toxicity. The neuroblasts formed in the matrix periventricular zones of the telencephalon in juvenile chum salmon are immature cellular forms that can express an incomplete set of glutamate NMDA receptors, migrating from the periventricular to the subventricular layers of the brain. In such undifferentiated cells, the cascade processes leading to apoptosis in mature neurons can be inhibited and, as a result, immature cells can avoid death and be successfully involved in the processes of neuronal regeneration.Experimental results have shown that the toxicity of glutamate during traumatic injury is attenuated by the action of Hchannels  and actichannels . However2S in the mammalian brain [2S production was observed after a traumatic injury.The insufficient supply of oxygen and glucose to the brain tissue in case of impaired blood circulation after a brain injury leads to a decrease in oxidative phosphorylation in neurons. As a result of reoxygenation, the amount of reactive oxygen species increases, which disrupts the functions of neuronal mitochondria and, as a result, further reduces the ATP production . Loss ofan brain , where a2S in the mammalian brain [2S production was observed after a traumatic injury. We assume that the increased production of H2S and GS in the telencephalon after the traumatic injury facilitates the reparative process. The increase in glutamate excitotoxicity after a telencephalic injury in O. keta can be neutralized by H2S, which reduces the toxic effects of glutamate. The H2S produced in the telencephalon can be considered as a proneurogenic and neuroprotective factor [2S production in the brain after injury should be explained from the point of view of maintaining cerebrovascular homeostasis, which implies antiapoptotic, anti-inflammatory, and antioxidant effects that reduce the level of secondary neuronal damage resulting from oxidative stress.Reparative neurogenesis in the mammalian brain is significantly hampered by the processes of secondary inflammation arising as a result of the toxic effects of glutamate and high excitotoxicity ,49,50,51an brain , where ae factor . Thus, tAs is known, Pax2 expression is regulated by the Shh signaling pathway ,55. HoweO. keta. This assumption is consistent with the data on the involvement of Pax2 in the process of regionalization of CNS in the embryonic and postembryonic periods of vertebrate development [The study on juvenile chum salmon has shown that Pax2 labels the population of neuroepithelial cells in different regions of the telencephalon. The expression of Pax2 in juvenile chum salmon was detected both in cell cytoplasm and in nuclei. Thus, in intact juvenile chum salmon the Pax2 labels a limited population of neuroepithelial cells and nuclei of unidentified cells, which, as we understand, participate in the processes of constitutive neurogenesis. Taking into account some data on the involvement of the PAX genes family in the construction of the spatial structure of CNS during embryogenesis and postembryonic development , we suggelopment .+ cells was typical for almost all regions of the telencephalon, which indicates the generalized expression of this TF. We identified the patterns of Pax2+ migrating cells in the area of injury, which is the final point of cell migration. Thus, after the injury to the telencephalon, we found that Pax2 expressing cells retain the function of spatial patterning of CNS, but its vector changes: in particular, instead of the population of resident NE cells, an intense Pax2 expression occurs in the population of migrating cells involved in the reparative reorganization of CNS. Nevertheless, the quantitative analysis showed a significant decrease in the number of Pax2+ cells in almost all areas of the pallial  and subpallial  zones of the juvenile chum salmon telencephalon  FEB RAS, the Resource Center of the NSCMB FEB RAS, and the Ethics Committee, which regulates the humane treatment of experimental animals .The material used in the work was 30 individuals of juvenile Animals were anesthetized in a cuvette containing 0.01% ethyl-3-aminobenzoate methanesulfonate (MS222)  for 5 min at room temperature. Using a sterile needle, a mechanical injury was applied to the region of the dorsolateral quadrant of the right hemisphere of the telencephalon to a depth of 1 mm. Immediately after the injury, the animals were released back into the tank for recovery and further monitoring.After the damaging effect in the telencephalon region, changes in the locomotor and behavioral activity in fish in the experimental group were monitored for 1 h. There were no significant changes in the locomotor activity in animals with the telencephalon injury compared with the control group. A small hematoma measuring 1–2 mm was clearly visible in the area of injury.At three days post-injury, all the experimental fish were deeply anesthetized in a 0.01% solution of MS 222  for 5 min to collect telencephalon samples. Animals were perfused with 4% paraformaldehyde solution  prepared in 0.1 M phosphate buffered saline  . After prefixation, the brain was removed from the cranial cavity and fixed in the same solution for 2 h at a temperature of 4 °C. Then, it was washed in a 30% sucrose solution at 4 °C for two days, with five changes of the solution. Serial frontal sections (50 μm thick) of the brain were cut on a freezing microtome .To study the localization of GS, CBS, and TF Pax2 in the telencephalon of juvenile chum salmon, immunoperoxidase labeling on frozen free-floating sections was used. Prior to immunostaining of the fish telencephalon tissue, the activity of endogenous peroxidase and nonspecific staining (background) was blocked. The endogenous peroxidase activity was blocked by incubation with 1% hydrogen peroxide for 20 min at room temperature. To eliminate nonspecific staining, the brain sections were incubated with non-immune horse serum. After incubation and washing with 0.1 M PBS, the frozen 50-μm brain sections were incubated in situ with primary rabbit anti-Pax2 polyclonal antibodies , rabbit anti-CBS polyclonal antibodies , and mouse monoclonal antibodies against GS  at 4 °C for 48 h. The anti-rabbit streptavidin–biotin imaging system  and the avidin–biotin (ABC) complex  were used for visualization of immunohistochemical (IHC) labeling products of polyclonal rabbit and monoclonal mouse antibodies, respectively. To identify the reaction products in both cases, a red substrate was used . Then, to identify immunonegative cells, the preparations were counterstained with a 0.1% methyl green (MG) solution . The color development was monitored under a microscope, washed with distilled water for 10 s, and then differentiated for 1–2 min in a 70% alcohol solution and then for 10 s in 96% ethanol. The preparations were dehydrated by a standard technique: they were kept in two changes of xylene, 15 min for each, and then placed under cover slips in the Bio-Optica medium .To assess the specificity of the immunohistochemical (IHC) reaction, a negative control method was used. The brain sections were incubated with a 1% solution of non-immune horse serum instead of primary antibodies for 1 day and processed as sections with primary antibodies. In all the control experiments, there was no immunopositive reaction.To visualize the IHC reaction in the telencephalon cells and conduct morphological analysis, we used a motorized microscope Axiovert 200 M equipped with a fluorescence ApoTome module and an attachment for improved contrast . The material was analyzed using the AxioVision software. Measurements were performed at 100×, 200×, 400× magnifications and in several randomly selected fields of view for each study area. The number of labeled cells in the field of view was counted at a magnification of 200×. Micrographs of the mounts were taken and analysis of the material was carried out using the Axio Vision software. The material was processed using the Axioimager program and the Corel Photo-Paint 14 graphics editor.The optical density (OD) of IHC labeling products in neuronal bodies and immunopositive granules was measured and the subsequent morphometric analysis of the parameters of cell bodies  was carried out using the software of the Axiovert 200 M microscope . For this, OD was measured at several sites of immunolabeled structures in a mount. Then, using the Wizard software, a standard OD assessment was carried out for intensely and moderately labeled and immunonegative cells. The averaged value of OD for each type of cells was subtracted from the mean value of OD of the background and, thus, the actual value of relative units of optical density (UOD) was obtained.O. keta, as a volumetric object in space, we used the stereological method of calculating the data obtained during microscopic analysis. For reliable spatial reconstruction for the ICH study, we used every 3 sections of the telencephalon. The stereological method enables, with the reliability determined by the objectives of the study and controlled by varying the parameters of the study design, the revealing of the morphometric characteristics of the object under study on the material of a limited number of sections. In this case, the systematic error (bias) is proven to be avoided, and the measurement error is controllable and directly depends on the sampling frequency: the more slices, the higher the accuracy. This is achieved through the use of an appropriate mathematical apparatus and adherence to sampling rules, in particular, a systematic random sampling.To obtain reliable quantitative characteristics of various regions of the telencephalon of juvenile When working with sections of the telencephalon, we selected the area under study, after which we took into account the morphometric parameters of interest. After receiving data from all selected slices, we performed calculations that allowed us to proceed to the description of the volumetric object. In particular, data were obtained on parameters such as the number of immunopositive cells within the dorsal, medial, lateral and central zones of the pallium and dorsal, ventral and lateral zones of subpallium, the density of distribution of immunopositive cells in constitutive and reactive neurogenic niches, and the number of immunopositive cells, nuclei and granules in the periventricular, subventricular and parenchymal zones of all parts of the telencephalon.Prior to the experiments, we performed a statistical analysis based on the variations in the measured parameters in our previous research  and detep < 0.05. The quantitative processing of morphometric data of IHC labeling was performed using the Statistica 12 and Microsoft Excel 2010 and STATA software packages . All variables measured in the groups were compared using one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls post hoc test. Comparison of the number of immunopositive cells between several neuroanatomical areas of pallium and subpallium of the intact or injured telencephalon (presented as groups) was performed using one-way analysis of variance (ANOVA) with Bonferroni’s correction. Values at p ≤ 0.05 and p ≤ 0.01 were considered statistically significant.The quantitative assessment of cells was carried out on a separately selected viewfield at a magnification of 10× of the objective and 20× of the eyepiece. All data analysis was performed using a blind test to reduce experimenter’s bias. The absolute number of cells of each type was counted. All quantitative results in the present study were analyzed with the SPSS software , and expressed as the mean ± standard deviation of the mean (M ± SD), and differences were considered statistically significant at + cells corresponds to the localization of CNN. On day 3 post-injury, significant changes in the GS localization were revealed in the telencephalon such as, in particular, the appearance of an additional type of GS+ neuronal precursors with an RG phenotype that are absent in intact juvenile chum salmon. The results of a comparative analysis indicate the predominance of RG in the pallial regions  in the post-traumatic period as compared to the subpallial regions . The maximum distribution density of RG in the pallium was found in DD and the minimum one in DL. After the traumatic injury to the chum salmon telencephalon, the patterns of CNN reactivation were identified; GS+ cells of both the neuroepithelial and RG types were found in the RNN.In the intact brain of juvenile chum salmon, GS labels a heterogeneous population of neuroepithelial-like cells localized in the PVZ, as well as a limited number of neuroepithelial type NPCs in the parenchyma. In the medial zone of the pallium (DM) and all subpallial zones, GS labeling was detected in extracellular granules located in the PVZ. In the subpallial area, the distribution of GS+ cells in all areas of the pallium and subpallium at 3 days after the telencephalon injury showed a significant increase in the number of CBS+ cells in all areas of the pallium  (p < 0.05) and the ventral area of the subpallium (BB) (p < 0.05), as well as in VL (p < 0.01). Thus, as a result of the telencephalon injury, the number of H2S-producing cells increased sharply, which caused an increase in the total H2S production in the juvenile chum salmon brain. Based on the fact that the processes of proliferation, cell migration, and neuronal differentiation in the PVZ intensify after a traumatic injury, we may consider H2S as a proneurogenic and neuroprotective factor contributing to the maintenance of cerebrovascular homeostasis and having the anti-inflammatory and antioxidant effects that reduce the level of secondary neuronal damage resulting from oxidative stress.In the dorsal and ventral parts of the juvenile chum salmon telencephalon, CBS labels a heterogeneous population of periventricular and parenchymal NE cells. In intact fish, the following common features of CBS localization were revealed: extended zones of distribution of intensely labeled cells alternating with areas devoid of immunopositivity, which corresponds to the patterns of GS distribution over NE cells in the intact telencephalon. After the damage to the telencephalon, the number of CBS-labeled cells of the NE type in the PVZ of the pallium and subpallium increased significantly compared to that in the control animals. The quantitative assessment of CBS+ cells migrating tangentially towards the injury area was identified in the PVZ. Thus, as a result of injury to the telencephalon of juvenile chum salmon, the function of spatial CNS patterning in Pax2+ cells is retained, but its vector changes; instead of the population of resident NE cells, intense expression of Pax2 was found in the population of migrating cells involved in reparative reorganization of CNS. The quantitative analysis showed a decrease in the number of Pax2+ cells in almost all areas of the pallial  and subpallial  parts of the telencephalon. We believe that the patterns of Pax2 expression in the juvenile chum salmon telencephalon on day 3 post-injury correspond to NPCs of both resident NE cells and reactivated trauma and are reduced as compared to those in intact fish. Further studies can clarify in detail the mechanism that we observed. However, we assume that the decrease in the Pax2 expression may be associated with the inhibitory effect of Pax6, with its expression, vice versa, increasing significantly after a traumatic injury.Pax2 labels the population of NE cells in different regions of the juvenile chum salmon telencephalon. The Pax2 expression was detected both in cell cytoplasm and in nuclei. Thus, in intact fish, the Pax2 protein product labels the gliogenic population of cells and nuclei of unknown functional specialization, which, as we believe, is involved in the processes of constitutive neurogenesis. After the brain injury, a population of Pax2+ neuroepithelial and glial precursors change as a result of injury to the telencephalon of juvenile chum salmon? What role does H2S play in the initiation of the acute inflammatory response in the brain of juvenile O. keta? Why does the number of Pax2+ cells in the telencephalon decrease in the acute post-traumatic period, while the number of Pax6+ cells increases? The answers to these questions can significantly extend the current knowledge of salmon neurogenesis as a convenient model for studying brain plasticity, reparation and postembryonic development.The obtained data raise various questions to address in the further study of neurogenesis in the adult Pacific salmon brain as follows. How does the ratio of GS


Objective: Bipolar disorders are among the most common chronic mental disorders. Despite the recent improvement in controlling psychiatric disorders, treatment of bipolar disorders remains a challenge. The aim of this study was to determine the effect of consuming probiotics in patients with bipolar disorder-type 1 compared to the placebo group.Method: This was a permuted blocked randomized clinical trial conducted in Shahid Beheshti mental hospital in Kerman, Iran, from October 2017 to October 2018. Two psychiatrists diagnosed and hospitalized all 38 patients with type 1 bipolar disorder based on the Diagnostic and Statistical Manual of Mental Disorders (DSM-5). Using blind randomized blocking method size 4, patients were divided into 2 groups of placebo and probiotic. Young Mania Rating Scale (YMRS) and Hamilton's Depression Rating Scale (HDRS) were completed at the beginning, week 4, and week 8 of the study by a psychiatry resident. Independent t test, Mann-Whitney and repeated measures ANOVA tests were used. Data were analyzed using SPSS software version 20. Results: There was no significant difference between the 2 groups in age, sex, and severity of mania and depression. Consumption of probiotics reduced the scores of YMRS and HDRS over time in the probiotic group more than the placebo group, but it was not significant.Conclusion: Consumption of probiotics had non-significant effects on improvement and treatment of bipolar type 1 patients. It is suggested that future studies be conducted with different probiotic microbial strains and longer period of treatment. The environment of the digestive system is the habitat of several microorganisms that help human health . DispropAlterations in the normal flora of the intestine have been shown in some of the brain-related disorders, eg, autism spectrum disorders , 6, multStudy of Pinto-Sanchez et al in 2017 showed that consumption of probiotic Bifidobacterium longum both reduces depression scores and alters brain activity and moderates response to fear stimuli in various brain regions . A similBipolar disorders (BD) are a leading global cause of disability  and chroStudy Procedure: This was a double-blind randomized controlled trial using placebo control that was performed at Shahid Beheshti hospital of Kerman Medical University. With census population sampling, all patients who met the inclusion criteria were enrolled in the study from October 2017 to October 2018. However, to estimate the high power of the study, the sample size was calculated based on the below formula: Considering standard deviation (δ) of 6, valuable group difference (d) of 6, type I error (α) of 0.05, type II error (β) of 0.1, and power of 90%, the calculated sample size was 21 patients in each group.Two psychiatrists diagnosed and hospitalized 38 male and female patients with type 1 bipolar disorder based on the Diagnostic and Statistical Manual of Mental Disorders (DSM-5). Study SampleThe inclusion criteria in the study, in addition to the diagnosis of type 1 bipolar disorder based on DSM-5 criteria, were age 18-65 years, not consuming any medication or discontinuing it within 2 weeks prior to the study, and not receiving ECT since 4 weeks prior to the study. The exclusion criteria were pregnancy and breast feeding, alcohol and drug use, suicide risk, use of probiotics and supplements over a period of 2 months before the start of the study, chronic diseases , active infection, schizophrenia, and other psychiatric disorders, and seizure, which were detected by 2 psychiatrists.Using permuted blocked randomized clinical trial, patients were divided into equal groups of placebo and probiotics with single-blinding in randomization. Allocation concealment was based on block size of 4. Patients were considered in blocks of 4 in their admissions in the hospital according to way A B A B  in randomization. Patients in both groups were allowed to receive lithium oxide, with a maximum dose of 900 mg per day, sodium valproate, with a maximum dose of 1200 mg per day, and, if necessary, risperidone. Probiotic group received a probiotic capsule , and placebo group received a placebo capsule of the same properties as probiotics in terms of color, size, and weight . Patients consumed the capsules daily and continued taking them after discharge. Drugs were kept in the refrigerator at the hospital and at home. Probiotics and placebo capsules were prepared by Tak-Gen-Zist . Duration of intervention was 8 weeks , and theStudy instrument: Mania Rating Scale (YMRS)-Persian version and Hamilton's Depression Rating Scale-Persian version with 17 items (HDRS-17) were used in this study.Young Mania Rating Scale (YMRS)This 11-item scale was developed by Young et al and is scored based on the Likert scale from zero  to 4 . . In the Hamilton's Depression Rating Scale (HDRS)This scale was developed by Hamilton to measure the severity of depression symptoms in a clinical interview. The scale has 24 items and is graded by a clinician based on the Likert scale. Its susceptibility to exacerbate depression cases was found to be 87%, and its sensitivity and specificity were found to be 62.4% and 92%, respectively . In IranEthical ConsiderationAll procedures of this study were based on the Helsinki Declaration. This study was approved by the Ethics Committee of Kerman University of Medical Sciences, with the ethical license number IR.KMU.AH.REC.1397.009, and at the same time it was registered in the Register of Clinical Trials, with registration number IRCT20180819040836N1. The intervention was explained to patients or their legal guardians and informed consent was obtained. Data AnalysisData were analyzed using SPSS version 20 software . The results were analyzed by independent t test, Mann-Whitney test, and Repeated Measure ANOVA test. P < 0.05 was considered statistically significant.In this study, 50 patients with bipolar disorder were hospitalized and selected. They were divided into 2 groups: probiotic (n = 25) and placebo (n = 25). During the first 4 weeks of the study, with a decrease of 6 in each group because of family consent to patient’s discharge despite doctor's advice and discontinuing the medication, the final sample in each group was reduced to 19. The mean age of the probiotic group was 38.9 years, with the standard deviation of 9.83, and that of the placebo group was 35 years, with the standard deviation of 8.18. There were no significant differences between the 2 groups in age and gender, and p value was 0.19 and 0.7, respectively.According to Table 1, probiotic consumption significantly decreased the scores of Young Mania  and Hamilton  questionnaire over time in bipolar type 1 recipient group receiving probiotics. Similarly, in the placebo group, the Young Mania  and Hamilton  questionnaire scores decreased over time in type 1 bipolar disorder group, and this difference was higher in the probiotic group than in the placebo group  & 2. There was no significant difference in the mania  and depression  scores between placebo and probiotics patients with type 1 bipolar disorder.In this randomized placebo-controlled study, the effect of taking probiotic supplements was evaluated on 38 patients with type 1 bipolar disorder. In this study, using the Hamilton’s depression and Young’s Mania Questionnaires at the beginning of the study (zero or baseline), 4 and 8 weeks after the onset of the intervention, the effects of probiotic consumption were evaluated on the severity of mania and depression in bipolar patients. The results of this study indicated that probiotic consumption did not significantly change the severity of depression and mania between the 2 study groups, meanwhile probiotic consumption significantly reduced the severity of depression and mania over time in the probiotic group.Inconsistent with this research, in the study of Akkasheh et al on 40 patients with MDD taking daily probiotics  for 8 weeks, significant reduction was found in depression symptoms . The ReiThe inconsistency of the results of this study with those of other studies may be due to the following reasons: the difference in sample size, bacterium’s type and strain, duration of study, the severity of the disease, and the simultaneous use of mood stabilizing drugs, dissimilarities in patient evaluation methods, geographical location of patients, and genetics. About the relationship between the strains of the probiotics and their efficacy on other diseases or disorders some studies were found. For instance, as some studies have suggested that only some probiotic strains have beneficial effects on IBS, diarrhea associated with antibiotics and H. pylori  infectioProbiotic supplementation as an adjuvant treatment in some mental disorders , 27 is aGlobal gut microbial community differences in BD showed decreased fractional representation of Faecalibacterium and an unclassified member from the Ruminococcaceae family, both from the phylum Firmicutes , while tIn this study, small sample size, the duration and the dose of probiotics consumed, and the possibility of interference between mood stabilizing drugs and the effects of probiotics could be cited as limitations.In addition, a number of patients did not continue consuming probiotics after discharge; therefore, the researchers had to select new patients from the community’s psychiatric center based on the study’s inclusion and exclusion criteria.According to the results of this study and based on the Hamilton’s Depression and Young Mania Scale, the use of probiotics mentioned in this study had no significant effect on improvement and treatment of patients group compared to placebo group. On the other hand, consumption of probiotics did not have a negative effect on patients and was completely tolerable for patients. Therefore, to better evaluate probiotics’ effects as a complementary treatment to decrease bipolar disorder severity, future studies should be conducted with different strains and increase the dose and duration of probiotic consumption. It is also recommended to evaluate cognitive functions and quality of life of patients and longevity in treatment (recurrence prevention).


Inter- and intra-batch variability of the quality attributes contribute to the uncertainty for demonstrating equivalent microstructure of post-approval changes and generic/hybrids of semisolid topical products. Selecting a representative sample size to describe accurately the in vitro properties of semisolids and to reach enough statistical power to demonstrate similarity between two semisolid topical products is currently challenging. The objective of this work is to establish the number of batches and units per batch to be compared based on different inter-batch and intra-batch variability to demonstrate equivalence in the physical characteristics of the products that ensure a similar microstructure of the semisolid. This investigation shows that the minimum number of batches to be compared of each product is 3 and the minimum number of units per batch could be 6 in the case of low intra- and inter-batch variability. If the products are not identical, i.e., 2.5–5% differences that are expected due to differences in the manufacturing process or the suppliers of excipients, 12 units and 6 batches are needed. If intra- or inter-batch variability is larger than 10%, the number of batches and/or the number of units needs to be increased. As the interplay between inter- and intra-batch variability is complex, the sample size required for each combination of inter- and intra-batch variability and expected difference between products can be obtained in the attached tables. The draft guideline on the quality and equivalence of topical products released by the European Medicines Agency (EMA)  states tThe demonstration of equivalence by in vitro testing, alone or in combinations with other studies, with at least 3 batches of test and 3 batches of reference is recommended also by the U.S. FDA in some product specific guidance for topical products , where a population bioequivalence approach is recommended for the comparison of relevant physical and chemical properties such as the particle size distribution of suspensions ,3,4,5,6.Sample size calculation is necessary for a correct demonstration of in vitro equivalence when a waiver of in vivo studies is applied. This comparability exercise has to be based on a protocol, where the objective of the comparison is defined , as well as the in vitro tests to be conducted to conclude in vitro similarity, the statistical methods to be employed in the comparisons, and the acceptance range, which is 10% by default in some of the EMA guidelines, unless otherwise justified ,10. UnfoRheological parameters, among others, are representative of the microstructure of topical products . The EMAIn a previous work ,15 all tThe objectives of this work are (i) to investigate the scenarios where the minimum sample size defined in the draft guideline is adequate and whether a smaller sample size might be acceptable in some cases, and (ii) to establish the number of batches and replicates per batch to be compared based on several scenarios of differences between test and reference products, inter-batch and intra-batch variability.T/R: Test/Reference ratio (%): 100 (no difference), 97.5 (2.5% difference) and 95 (5% difference)B: Number of batches per product: 1, 2, 3, 6 and 12NU: Number of units/replicates per batch: 6, 12 and 24NIBV: Inter-batch variability (%): 0, 2.5, 5, 7.5, 10, 15 and 20ABV: Intra-batch variability (%): 0, 2.5, 5, 7.5, 10, 15 and 20The simulated scenarios were generated from the combination of the different variables specified below:B , 3 levels of NU , 7 levels of IBV , and 7 levels of ABV . All study design conditions were arbitrary selected, but they were aimed to describe frequently observed experimental conditions. In addition, some of these scenarios provided the same total number of observations  to assess the better way to address the inter- and the intra-batch variability. The inter-batch and intra-batch variability encompass both low and high variability conditions observed in the literature [A total of 2205 scenarios were simulated, assuming 3 levels of T/R, 5 levels of Nterature ,17,18,19R) was fixed to a value of 1 and the population value for the test product (TVPT) was established based on the T/R ratio considered in the simulation settings.The simulation of a single experiment started with obtaining the population parameters of each product. The population value of the reference product  with replacement was conducted on each experiment per scenario based on the simulated data in order establish the bootstrap distribution of the parameter for each experiment of each scenario. Ninety percent non-parametric confidence intervals were calculated from the bootstrap distribution of the geometric mean ratios using the percentile method. Given the bootstrap distribution of the parameters, θ(*1), θ(*2),…,θ(*B), the percentile confidence interval is the interval between the 100·α and 100·(1 − α) quantiles of the bootstrap distribution, where α is the nominal type I error. Similarity was concluded when the non-parametric 90% confidence interval for the ratio test/reference was within the ±10% acceptance range (90.00–111.11%).A non-parametric bootstrap  and R-studio  were used for the bootstrap analyses and graphical display, respectively. All the analyses were performed on a Linux server from the University of Valencia (LluisVives) with Xeon 7500 series hexacore at 2.67 GHz and 2048 GB RAM.R , and secondly, the estimation of the probability of showing equivalence through the stochastic simulation of 1000 experiments per scenario, including 10,000 bootstraps on each simulation in order to calculate the 90% CI of the T/R ratio.B, NU, IBV and ABV. These tables include the possibility of zero inter- and intra-batch variability, although not realistic, to show that bioequivalence is always shown in the absence of variability since the simulated differences are lower than 10%. These scenarios might represent scenarios with extremely low variability (<0.5%). In such cases, the sample size  could be extremely low.These simulations show that with the conditions required in the draft guideline (3 batches and 12 replicates), there is statistical power to conclude similarity only if the IBV and ABV does not exceed 7.5% if the products are equal, and 5% if the products are 2.5 or 5% different, which a usual assumption when calculating the sample size. If the IBV is low  there is at least 80% power in scenarios of more ABV . Similarly, if the ABV is only 5%, there is at least 80% power to show similarity for IBV of 7.5% when the products differ 2.5%, but 5% IBV if the products differ 5% in their mean values. Therefore, the number of batches or the number of replicates have to be increased when the variabilities are higher than 7.5%, or even 5% if the products are not identical.n = 10,000) for each scenario, assuming a T/R = 100% in the rheological parameter between test and reference products and stratified by NB and NU are depicted in The distribution of the lower and upper limits of the 90% CI derived from the bootstrap analysis  are assessed , while if the sample is increased (6–12 batches), a more precise characterization of the variability can be obtained. This trend is independent of the Nassessed . As long and ±5% .This investigation provides the number of batches and the number of replicates per batch required to have enough (>80%) statistical power to show equivalence within a 10% acceptance range where multiple batches are included in the comparison , in contrast to those comparisons where only batch is tested . In the case of in vivo studies, it is assumed that the in vivo response  would be the same in all manufactured batches, therefore only one batch needs to be tested, but for in vitro parameters  several batches need to be tested because the in vitro outcome varies from batch to batch and between units of a given batch. Importantly, this investigation is not limited to in vitro properties of semisolid dosage forms, but can be applied to any comparison of in vitro parameters that can be assumed to follow a normal/log-normal distribution and where several batches of each product are tested and more than 1 determination is conducted per batch.In addition, this study also addresses the adequacy of the frequent requirement of testing 3 batches and 12 replicates per batch. As can be seen in Furthermore, the comparison of the combination of batches and replicates that produce the same total number of observations , shows that 12 batches and 6 replicates are successful in a larger number of scenarios independently of the difference between products. A general recommendation for moderate-to-high variability (≥10%) scenarios (IBV and/or ABV) could be to use 6 batches and 12 units per batch or, in case there is not sufficient number of batches available, 3 batches with 24 units per batch.The evaluation of the probability of showing equivalence where it is not possible to use three batches because fewer batches are on the market shows that the use of two batches is surprisingly counter-productive. The use of only one batch is impractical, unless the inter-batch variability is known to be very low through the investigation of multiple batches during the pharmaceutical development, since testing only one batch does not allow the characterization of the inter-batch variability. The obtained estimations of the mean would be only accurate if the inter-batch variability were extremely low. When the variability is so low any selected batch can be considered to be representative of future batches to be manufactured for the test product and for any other batch of the reference product in the market. This same assumption is employed when conducting in vivo bioequivalence studies, where it is expected that all batches of the same manufacturer have consistently the same bioavailability. Testing only one batch of test and reference in products with known low inter-batch variability (<2.5%) could be feasible if a large number of replicates per batch were tested. In case of 24 replicates, enough power is achieved for scenarios with 2.5% inter-batch variability only and intra-batch variability of 10% if the ratio between products is 100%, 7.5% intra-batch variability if the ratio is 97.5%, and 5% if the ratio is 95%.This study also confirms the fact that increases in IBV must be addressed by increasing the number of batches. This trend is irrespective of the T/R ratio simulated  and the number of units  considered, but only visible when an appropriate characterization of the IBV occurs . For example, in case of identical products or differences of 2.5% or 5%, and ABV of up to 10%, the increase of IBV from 5 to 10% requires testing 6 instead of 3 batches to achieve 80% power to show similarity. This conclusion is supported in R, where the limits start to be widened, and S0, which defines the scaling proportionality constant, are the same in the Guideline on the investigation of bioequivalence [ivalence . If it wivalence . FurtherThe magnitude of the IBV and ABV as well as the difference between test and reference differ in the multiple in vitro parameters under comparison. For this reason, the calculation of sample size could be based either individually for each parameter according to the expected variability and the expected difference between products for that parameter, or globally for all parameters based on the maximum sample size required for the parameter that presents greater variability and/or difference. Those decisions can be conditioned by the cost of the analysis and the availability of batches and units. Additionally, as the sponsor has to compare multiple in vitro parameters, multiplicity may cause that some comparison may be non-equivalent simply by chance. The higher number of in vitro parameters, the higher probability to conclude that one of them is not able to show equivalence. Therefore, it would be wise to use the maximum global sample size if affordable. In addition, the multiplicity can be compensated with the possibility to repeat the studies with a higher sample size to achieve conclusive results. In such case, the whole evidence from inconclusive and conclusive studies should be combined to demonstrate consistency.In conclusion, this simulation-based analysis shows the influence of inter- and intra-batch variability on the sample size required to conclude on the equivalence between the in vitro characteristics assuming test/reference ratios of 100%, 97.5% and 95%. This work confirms that the influence of inter-batch variability should be addressed by increasing the number of batches and the intra-batch variability could be solved by increasing the number of units/replicates per batch. Testing 3 batches with 12 units per batch seems to be insufficient unless the inter-batch variability is known to be low. Testing 6 batches with 12 units per batch or 3 batches with 24 units per batch would be sufficient to declare the equivalence in most variability scenarios with up to 5% difference between test and reference products.


The peak shape analysis of the X-ray diffraction patterns, the EBSD observations, and the EDS spectra of the alloys with different compositions show that the characteristics of the dendrites and the hard phase, Cr23C6, into the initial single-phase face-centered cubic (FCC) matrix becomes gradually visible as the carbon content increases from 0 to 4%. The crystal phase variations lead to a non-linear orientation of the microstructure, to a refinement of the grains, and to a higher elastic modulus. This study presents the solid saturation limit of the interstitial carbon atoms in such alloys and establishes an empirical relation between an alloy’s elastic modulus and its carbon content.In this paper, a (Fe The effects of the interstitial carbon atoms on its microstructure and mechanical properties were studied to provide guidance for the development of such metallic composites.As a kind of super-solid solution different from the traditional alloys , high-enFe, Mn, Co, and Cr powders were weighed and mixed according to a mass ratio of 5:3:1:1 . The graphite powder was sequentially weighed and incorporated into the mixed metal powder in different mass fractions  to obtain the starting material of the alloys . These samples were weighed separately, placed in a powder pressing instrument, and pressed into a block under a load of 5 KPa. The block was washed with ethanol in an ultrasonic bath and dried. Then, it was placed into a copper crucible in a vacuum arc smelting furnace, which was initially evacuated and then filled with argon gas. The arc was ignited (the maximum melting current was maintained below 800 A) and the alloy was melted. Via electromagnetic stirring, the molten alloy solution was stirred uniformly and the forward and reverse melting were carried out repeatedly to obtain an ingot, as shown in w(C)%, reaches 4%, the characteristic peak of the cementite appears, indicating its formation. One can speculate that the carbon atoms reach their solution limit in the voids of the FCC lattice when the solid solubility is 4%. As the carbon content continues to increase, the intensity of the main peak (200) in the FCC decreases, reflecting the precipitation of the carbides.w(C)% smaller that 4% show a similar average matrix grain sizes (about 95 μm) upon performing the calculations based on the linear intercept method [w(C)% higher than 4% present obvious characteristics of dendrites and carbides % is lower than 4%. When the w(C)% is higher than 4%, the carbon atoms in the matrix begin to saturate and the excessive carbon atoms react with the matrix to form Cr23C6, which starts to precipitate. The distribution of Cr23C6 at the grain boundary of the matrix directly leads to a decrease in the grain size of the as-cast alloy. Moreover, as shown in the histogram (w(C)% increases from 2% to 4% and this is probably due to the low amount of dendrites in the alloy. However, the grain orientation of the entire system become observably equiaxed when the w(C)% reaches 8%, reflecting the effect of the carbides, which are formed at the grain boundaries.The EBSD orientation profile and the histogram shown in es about 5 μm uponcarbides . An elemistogram , the oriw(C)% on the alloy’s mechanical properties was studied by converting the load-indentation depth data measured via the nanoindentation technique (w(C)%, and the elastic modulus, E, which can be defined by the following mathematical expression: E = 183.4+24.5×w(C)%×100. This linear positive correlation between E and w(C)% is attributed to the combination of the dispersion-strengthening effect of the generated Cr23C6 when compared to the matrix. For the Young’s Modulus stagnation at approximate 400 GPa, it is supposed to attributed to following reasons: 1) carbon saturation rate is around 6%wt level, so after this threshold, the carbides generated and located at the grain boundaries. This caused a huge jump for elastic modulus comparing to previous samples. 2) after large amount of carbides generation, the carbide is not the main variable to affect the overall mechanical strength, so then stagnation from 6%wt to 10%wt samples at 400GPa was observed. Moreover, the strengthening, due to the grain size reduction of Cr23C6, inhibits the grain growth of the as-cast HEAs matrix. Although the hard phase (Cr23C6) with a random FCC structure [The effect of the echnique  into an echnique . These rThe solid solubility of the interstitial carbon atoms in the matrix lattice of a FeMnCoCr alloy measures 4%.23C6 formed by the presence of C and Cr in the HEAs can modify the grain orientation of the alloy system.The increase in the quantity of CrE, and the carbon content, w(C)%, exhibits an approximately linear positive correlation.The elastic modulus,


There is a high level of sodium fluoride in SPL-1cut—about 15%—and sodium fluoride is randomly distributed in the carbon granule. The recycling of SPL-1cut using dolomite as a reactant, based on the characteristics of the two-step decomposition of dolomite at a high temperature, is discussed. The recycling of SPL-1cut was performed under the following heating conditions: the heating temperature was 850 °C, the holding time was 120 min, and 40% of the dolomite was added to the SPL-1cut. It was found that the cyanides are completely oxidized and decomposed, and dolomite is decomposed into MgO and active CaCO3. At the same time, NaF reacts with active CaCO3 and converts into CaF2. The results provide references for using SPL-1cut as an alternative fuel in the dolomite calcination process of the Pidgeon Process.Spent Pot Lining First Cut (shortened to SPL-1cut) is a solid waste discharged from a primary aluminum electrolytic production process. SPL-1cut is classified as hazardous waste in China because it contains large amounts of soluble sodium fluoride and a tiny amount of cyanide. Most of SPL-1cut is carbon—about 65%—and its calorific value is 22.587 MJ∙kg The alumina-cryolite molten salt electrolysis process is currently the most mature industrial-scale primary aluminum production method and is widely used throughout the world. The production of primary aluminum from molten salt electrolysis has brought about some environmental problems, among which spent pot lining (SPL) is one of the most prominent. The service life of aluminum electrolytic cells is generally 5–8 years ,2. About3. Heating at a high temperature is the simplest and most effective way to eliminate cyanides, which begin to decompose when heated to above 400 °C [3 in a high-temperature environment, the soluble NaF in SPL can be converted into stable CaF2, reducing the harm of soluble fluoride sharply. In previous studies, limestone was used to convert harmful fluorides into harmless and stable fluorides [The toxicity of SPL-1cut is mainly due to soluble fluorides and cyanides. The content of fluorides in SPL-1cut is relatively high, and the most common type of them are sodium fluoride. Sodium fluoride is easily soluble in water, causing serious pollution to water resources and soils and easily forming high-fluorine water and high-fluorine soil, which in turn endangers the growth and survival of animals and plants. When cyanides meet water, they will release the harmful gas NHe 400 °C ,12. In gluorides ,13,14. H3)2. Dolomite, after crushing and calcining, is an important raw material for the silicon thermal process of magnesium (the Pidgeon Process). MgCO3 and CaCO3 in dolomite exists in the structure of double salt with a strong binding force [3 in limestone. It is interesting that dolomite has the characteristics of two-step decomposition at high temperatures and fluorite is used as a catalyst in the Pidgeon Process [2 is mainly derived from natural fluorite, which is scarce in China. The use of natural fluorite is one of the reasons for the high cost of the Pidgeon Process. Based on the characteristics of the SPL-1cut and dolomite, some interesting experiments are put forward. What will happen if a certain amount of dolomite is added to the SPL-1cut at a high temperature? The following experimental results are expected: CaCO3 in the dolomite can convert fluorides into CaF2; toxic and harmful substances such as cyanides, nitrides, and carbides in the SPL-1cut can be eliminated at high temperatures; at the same time, the carbon in the SPL-1cut is fully burned at high temperatures to generate heat and provide reaction energy.Dolomite is a kind of natural ore, and its main component is CaMg and fully mixed according to the mass ratio of 1:1:1, and then the mixed sample was ground by a ball mill and stored in an airtight plastic bucket for subsequent tests and experiments. X-Ray Diffractomer (XRD) , scanning electron microscope (SEM) , a calorimeter , and a sulfur analyzer  were used for identifying the physical and chemical properties of SPL-1cut. The phase composition of SPL-1cut is shown in The matrix of the SPL-1cut is carbon and contains sodium fluoride, calcium fluoride, alumina, cryolite, and other components  and a ball mill  to obtain a suitable powder. The chemical components of dolomite are shown in SPL-1cut and dolomite were crushed and sieved separately, mixed according to a certain ratio, and loaded into corundum crucibles , then crucibles with the experimental sample were placed in an ultra-high-temperature muffle furnace  for heat treatment. The purpose of the experiments was mainly to eliminate cyanides in SPL-1cut and to obtain a high conversion ratio of fluoride. The experimental temperature was 750–950 °C, the particle size of the sample was in the range of 0–200 mesh, the residence time was 90–210 min, and the mass ratio of dolomite in the experimental sample was from 10 to 50 wt%. The influences of the temperature, the particle size of the sample, the residence time, and the weight ratio of dolomite on the reaction loss ratio were studied.The main reactions during the heat treatment were the combustion of carbon and the reactions between fluoride and dolomite .3, which helps promote the conversion of fluoride.The combustion of carbon not only causes a large loss of the total weight of the sample but also causes more closer contact between fluoride and CaCO1 is the initial weight of the mixed sample and g2 is the weight of the final product after processing.In order to identify which condition is better, the reaction loss ratio Y is used to visually judge the experimental results.0 as the optimal loss ratio, which only includes the carbon burning loss and the decomposition loss of magnesium carbonate in dolomite during the thermal treatment; YMAX is the maximum loss ratio, which is the complete combustion loss ratio of SPL-1cut plus the complete decomposition loss ratio of dolomite. SPL-1cut did not react with the dolomite during the process.1 is the weight fraction of SPL-1cut in the mixed sample, c2 is the weight fraction of dolomite in the mixed sample, Y1 is the loss ratio when SPL-1cut is separately processed, Y2 is the loss ratio when dolomite is separately processed, and Y3 is the loss ratio when the first decomposition step of dolomite occurs—that is, only the MgCO3 in dolomite is completely decomposed into MgO.Different experiments were carried out under different experimental conditions according to the preset experimental scheme. Experimental results are shown in 2CO3 in the reaction product is 851 °C. At 900 °C, Na2CO3 easily melts and the mixture becomes sticky, causing a small decrease in the loss ratio. The addition of dolomite achieves the purpose of converting fluoride into CaF2, and prevents a large increase in the loss ratio when the temperature is up to 950 °C. However, due to the inadequate amount of dolomite, the fluoride was not completely converted.It can be seen clearly from The loss ratio of the experimental sample becomes larger and larger when the particle size of the sample becomes finer and finer. The maximum value can be obtained when the particle size of the sample is finer than 150 mesh. As the particle size continues to become finer than 200 mesh, the loss ratio significantly decreases  EDS analysis of different positions of the final product (wt%).(2) EDS analysis of different positions of the final product (At%).3·CaCO3 in dolomite exists in a double salt form whose high-temperature decomposition process can be divided into two steps. The decomposition of MgCO3 occurs first, and then CaCO3 decomposition occurs. This characteristic of dolomite ensures that it can provide effective, active CaCO3 as a reactant when reacting with fluorides in SPL-1cut. It can be clearly seen from 2 and MgO. In 3 and NaF; area b is a complete conversion place of fluoride, and its main substances are CaF2 and Na2CO3; the main substances in area c are MgO and NaF, and the first step of dolomite decomposition occurred in this area. During thermal processing, MgCO3·CaCO3 in dolomite was decomposed to generate MgO and CaCO3, and the newly generated CaCO3 reacted with NaF to ensure the conversion of fluorides. Furthermore, along with the temperature rises, carbon combustion, and dolomite first-step decomposition, reactions between fluorides and calcium carbonate occur in turn. When dolomite is used to treat SPL-1cut, the final product containing MgO and CaF2 can be more easily recycled in the Pidgeon Process. The process has more practical value compared with the previous process, which used limestone. MgCO3. At the same time, the MgCO3 in dolomite was decomposed into MgO. In a word, the processing of SPL-1cut is feasible by adding dolomite. The process has the following advantages: the temperature is not high, the concentration of soluble fluoride in the final product can be reduced to a safe limit, and the final product does not show an obvious adhesion phenomenon.When the SPL-1cut was processed for 120 min under the conditions of 850 °C, particle size finer than 100 mesh, and a dolomite addition fraction at 40 wt%, the complete conversion of fluorides was achieved by fluorides reacting with newly generated CaCOThe leaching toxicity of the SPL-1cut final product (treated with dolomite) was measured, and the results are listed in − and CN− in the final product are lower than the national limit (GB5085.3-2007) [It can be seen from .3-2007) . 3 newly generated by the first-step decomposition of dolomite will react with fluorides to generate calcium fluoride, an insoluble fluoride. The cyanides will be completely oxidized and decomposed. The exothermic combustion of carbon provides enough heat for other reactions.The recycling of SPL-1cut is achieved by adding dolomite to SPL-1cut based on the characteristics of the two-step decomposition of dolomite at high temperatures. During the thermal process, the CaCO2. It can be used as a raw material and as an alternative fuel for magnesium production by the Pidgeon Process so as to save fluorite and standard coal.Dolomite is used as a reactant to reduce the soluble fluorides in SPL-1cut. The final product is mainly composed of MgO and CaFThe recycling feasibility of SPL-1cut in magnesium production by the silicon thermal process is discussed in this paper, and some preliminary results were obtained. In the future, continuous research will be conducted on the following aspects: the addition of SPL-1cut instead of standard coal for the dolomite calcination process, including the blending ratio of SPL-1cut, and the influences of SPL-1cut on the magnesium reduction tank and magnesium yield.