In Situ Hybridization Histochemistry Research Articles

Long non-coding RNA AK001903 regulates tumor progression in cervical cancer.

A majority of cervical cancers are squamous cell carcinomas, arising from the squamous (flattened) epithelial cells that line the cervix. Long noncoding RNAs (lncRNAs) are a unique class of messenger RNA-like transcripts of at least 200 nucleotides in length with no significant protein-coding capacity. Aberrant lncRNA expression is emerging as a major component of the cancer transcriptome. In the present study, lncRNA microarrays were conducted to investigate the differentially expression lncRNAs in cervical cancer (CC) tissues compared with peritumoral tissues. Then, the most significantly upregulated lncRNA, which was lncRNA-AK001903 was selected to conduct further experiments. Real-time Quantitative polymerase chain reaction was conducted to investigate lncRNA-AK001903 expression in CC tissues and Hela, Siha, Ca Ski, C33a, H8 (HPV-immortalized cervical epithelial cell line) cell lines, and in situ hybridization histochemistry (ISHH) was performed to detect lncRNA-AK001903 expression level in different CC stages. The effect of lncRNA-AK001903 on cell proliferation, invasion and migration was assessed after knockdown of lncRNA-AK001903. The findings of the study confirmed that lncRNA-AK001903 was upregulated in CC cells and tissues compared with normal cell line H8 and peritumoral tissues. ISHH demonstrated that the expression level of lncRNA-AK001903 was connected with International Federation of Gynecology and Obstetrics (2018) stage of CC. Knockdown of lncRNA-AK001903 inhibited cell proliferation, invasion and migration in Ca Ski cells. In conclusion, lncRNA-AK001903 was demonstrated to be an oncogenic lncRNA that promotes tumor progression and may be an effective target for CC treatment in the near future.

Increased Dopamine Type 2 Gene Expression in the Dorsal Striatum in Individuals With Autism Spectrum Disorder Suggests Alterations in Indirect Pathway Signaling and Circuitry.

Autism spectrum disorder (ASD) is behaviorally defined and diagnosed by delayed and/or impeded language, stereotyped repetitive behaviors, and difficulties with social interactions. Additionally, there are disruptions in motor processing, which includes the intent to execute movements, interrupted/inhibited action chain sequences, impaired execution of speech, and repetitive motor behaviors. Cortical loops through basal ganglia (BG) structures are known to play critical roles in the typical functioning of these actions. Specifically, corticostriate projections to the dorsal striatum (caudate and putamen) convey abundant input from motor, cognitive and limbic cortices and subsequently project to other BG structures. Excitatory dopamine (DA) type 1 receptors are predominantly expressed on GABAergic medium spiny neurons (MSNs) in the dorsal striatum as part of the “direct pathway” to GPi and SNpr whereas inhibitory DA type 2 receptors are predominantly expressed on MSNs that primarily project to GPe. This study aimed to better understand how this circuitry may be altered in ASD, especially concerning the neurochemical modulation of GABAergic MSNs within the two major BG pathways. We utilized two classical methods to analyze the postmortem BG in ASD in comparison to neurotypical cases: ligand binding autoradiography to quantify densities of GABA-A, GABA-B, 5-HT2, and DA type 1 and 2 receptors and in situ hybridization histochemistry (ISHH) to quantify mRNA for D1, D2 receptors and three key GABAergic subunits (α1, β2, and γ2), as well as the GABA synthesizing enzymes (GAD65/67). Results demonstrated significant increases in D2 mRNA within MSNs in both the caudate and putamen, which was further verified by proenkephalin mRNA that is co-expressed with the D2 receptor in the indirect pathway MSNs. In contrast, all other GABAergic, serotonergic and dopaminergic markers in the dorsal striatum had comparable labeling densities. These results indicate alterations in the indirect pathway of the BG, with possible implications for the execution of competing motor programs and E/I imbalance in the direct/indirect motor feedback pathways through thalamic and motor cortical areas. Results also provide insights regarding the efficacy of FDA-approved drugs used to treat individuals with ASD acting on specific DA and 5-HT receptor subtypes.

A preliminary investigation of phoshodiesterase 7 inhibitor VP3.15 as therapeutic agent for the treatment of experimental autoimmune encephalomyelitis mice

cAMP plays a significant role in signal transduction pathways controlling multiple cellular processes such as inflammation and immune regulation. cAMP levels are regulated by a family of phosphodiesterases (PDEs). We have studied the effects of a novel PDE7 inhibitor (PDE7i) treatment on mice with experimental autoimmune encephalomyelitis (EAE) a model of multiple sclerosis (MS) and compared it with another PDE7i. EAE was induced by immunizing C57BL/6J mice with myelin oligodendrocyte glycoprotein (MOG35-55) peptide. Mice were treated daily either from disease onset or from disease peak with each PDE7i and with fingolimod (used in therapy for MS patients) and disease evolution was followed by clinical symptoms. We examined neuropathology of spinal cord, ex vivo lymphocyte proliferation by [3H]-thymidine incorporation, TNFα by ELISA and cAMP-PDE mRNAs expression by in situ hybridization histochemistry (ISHH) in spinal cord of EAE mice treated with both PDE7 inhibitors. Treatment of EAE mice with the novel PDE7i, VP3.15 showed more efficacy in reducing clinical signs at 10mgkg−1 than the other PDE7i, BRL50481 and similar to fingolimod. VP3.15 acts on peripheral lymphocytes inhibiting their proliferation and TNFα secretion in a dose-dependent manner. PDE7i treatment alters the levels of PDE4B and PDE7 mRNA expression in EAE mice spinal cord. Given the interest in the development of new drugs for MS, including PDE7i as anti-inflammatory drugs, it is important to study the role played by PDE7 in neurodegenerative diseases with inflammatory component to better understand the beneficial and detrimental effects of a future therapy.

Abstract 2784: Targeting TGF-β regulated E3 ligases: a novel therapeutic approach for primary liver cancer

Abstract Background: Hepatocellular cancer (HCC) is the 3rd leading cause of cancer death in the world, that is difficult to treat due to late diagnosis and few viable targeted therapeutics. A large number of studies have demonstrated that the TGF-β pathway plays a fundamental role in the biology of the GI tract, and as such, several components of the pathway are commonly targeted in various GI cancers. TGF-β signaling is regulated by the ubiquitin-proteasome pathway, in which E3 ubiquitin ligases recognize and target proteins for degradation by the proteasome. Numerous E3 ubiquitin ligases have been identified as negative regulators of different components of the TGF-β pathway. More recently, Praja and Keap1 have been identified as E3 ligases that ubiquitinates β2-spectrin (β2SP) in a TGF-β-dependent manner. Accordingly, Praja E3 ligase activity regulates TGF-β signaling by controlling β2SP abundance through ubiquitin-mediated degradation. Hypothesis: Specific E3 ligases, which disrupt TGF-β/β2SP/Smad3 tumor suppressor pathway lead to uncontrolled activation of chromatin and tumor formation. Therefore, small molecule inhibitors that specifically target these E3 ligases could present a novel therapeutic approach for liver cancer. Materials & Methods: Broad genome datasets including 110 cases of HCC patients were retrieved from The Cancer Genome Atlas (TCGA) and analyzed. 23 HCC and normal liver tissue specimens were immunostained with anti-Praja1 antibody. Whole mount in situ hybridization histochemistry (ISHH) assay was used to determine knock down Praja1in zebrafish embryos. A small chemical compound library was screened for E3 ligase inhibitors targeting Praja1. Results: (1) Broad genome analysis reveals that E3 ubiquitin ligases regulating the TGF-β pathway are altered in GI cancers mainly by increase in mRNA and that they negatively correlate with patient survival. (2) Praja1 expression is dramatically raised in human HCCs with loss of TGF-β signaling. (3) Praja1 inhibits TGF-β/Smad3 tumor suppressor function and increases c-Myc activities. (4) Inhibition of Praja1 leads to apoptosis and suppresses its oncogenic activities. (5) Small chemical inhibitors suppress Praja1 activities and restores TGF-β tumor suppressor function in HCC cells. (6) Small chemical inhibitors induce apoptosis and inhibit HCC cell growth and tumorigenesis. Conclusions: E3 ubiquitin ligases that modulate the TGF-β pathway are frequently increased in HCC. Praja1 presents a therapeutic target- it disrupts the TGF-β tumor suppressor pathway, and its overexpression promotes cancer cell survival and proliferation. Small molecule inhibitors such as triterpenoids that specifically target Praja1 could be very useful in HCC therapy, through targeting c-Myc, restoring TGF-β tumor suppressor function. This study may lead to new therapeutics targeting this lethal cancer and potentially a Phase I clinical trial in HCC. Citation Format: Jian Chen, Jiun-Sheng Chen, YoungJin Gi, Lior H Katz, Ji-Hyun Shin, Liem Phan, Wilma Jogunoori, Vivek Shukla, Bibhuti Mishra, Shulin Li, Milind Javle, Mien-Chie Hung, Lopa Mishra. Targeting TGF-β regulated E3 ligases: a novel therapeutic approach for primary liver cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2784. doi:10.1158/1538-7445.AM2014-2784

CX3CL1 (fractalkine) protein expression in normal and degenerating mouse retina: in vivo studies.

We aimed to investigate fractalkine (CX3CL1) protein expression in wild type (wt) retina and its alterations during retinal degeneration in mouse model (rd10) of retinitis pigmentosa. Forms of retinal protein CX3CL1, total protein and mRNA levels of CX3CL1 were analyzed at postnatal days (P) 5, 10, 14, 22, 30, 45, and 60 by Western blotting and real-time PCR. Cellular sources of CX3CL1 were investigated by in situ hybridization histochemistry (ISH) and using transgenic (CX3CL1cherry) mice. The immunoblots revealed that in both, wt and rd10 retinas, a membrane integrated ∼100 kDa CX3CL1 form and a cleaved ∼85 kDa CX3CL1 form were present at P5. At P10, accumulation of another presumably intra-neuronal ∼95 kDa form and a decrease in the ∼85-kDa form were observed. From P14, a ∼95 kDa form became principal in wt retina, while in rd10 retinas a soluble ∼85 kDa form increased at P45 and P60. In comparison, retinas of rd10 mice had significantly lower levels of total CX3CL1 protein (from P10 onwards) and lower CX3CL1 mRNA levels (from P14), even before the onset of primary rod degeneration. ISH and mCherry reporter fluorescence showed neurons in the inner retina layers as principal sites of CX3CL1 synthesis both in wt and rd10 retinas. In conclusion, our results demonstrate that CX3CL1 has a distinctive course of expression and functional regulation in rd10 retina starting at P10. The biological activity of CX3CL1 is regulated by conversion of a membrane integrated to a soluble form during neurogenesis and in response to pathologic changes in the adult retinal milieu. Viable mature neurons in the inner retina likely exhibit a dynamic intracellular storage depot of CX3CL1.

Abstract 5198: Targeting E3 ligase PRAJA1 in hepatocellular cancer .

Abstract Background: Hepatocellular Cancer (HCC) is a rising and lethal disease, that is difficult to treat due to late diagnosis and few viable targeted therapeutics. Recent studies support a key role for TGF-β signaling in suppressing these tumors. Expression of the E3 ubiquitin ligase PRAJA, a negative regulator of TGF-β signaling via Smad3 and its adaptor β2-spectrin (β2SP), is dramatically raised in up to 70% of human HCCs. This indicates that PRAJA1 overexpression suppresses Smad3 and β2SP mediated TGF-β tumor suppressor signaling hence promoting HCC progression. Hypothesis: Disruption of TGF-β/β2SP tumor suppressor pathway by PRAJA1 leads to uncontrolled activation of chromatin and tumor formation. Therefore, small molecule inhibitors that specifically target PRAJA1 could be very useful in HCC therapy. Materials & Methods: Cancer patient datasets were retrieved from Gene Expression Omnibus and analyzed with Oncomine analysis tools. We analyzed 3 group-arrays of liver tumor tissues (total 298 cancer tissues vs. 249 normal tissues, p-value<1E-4). We also analyzed 55 arrays for other cancers (total 2850 cancer tissues vs. 779 normal tissues, p-value<1E-4). To validate protein expression, specimens from 13 HCC and normal liver tissues were immunostained with anti-PRAJA1. Whole mount in situ hybridization histochemistry (ISHH) assay was used to determine knock down PRAJA1in zebrafish embryos. Soft agar assay and colony formation assay were performed to elucidate PRAJA1 oncogenic activity in HCC cells. Additionally, we screened a compound library for E3 ligase inhibitors targeting PRAJA1. Results: (1) PRAJA1 expression is dramatically raised in human HCCs with loss of TGF-β signaling. (2) PRAJA1 interacts with β2SP/Smad3 and inhibits TGF-β tumor suppressor target gene activation. (3) Inhibition of PRAJA1 in the developing Zebrafish embryo and HCCs leads to high levels of apoptosis. (4) Overexpression of PRAJA1 leads to increased HCC growth; conversely knock down of PRAJA1 suppresses its oncogenic activities. (5) TGF-β promotes complex of CTCF and β2SP in nucleus. (6) RTA402/RTA405 inhibits PRAJA1 and restores TGF-β tumor suppressor function in HCC cells. (7) RTA402/RTA405 induces apoptosis and inhibits HCC cell growth and tumorigenesis. Conclusions: PRAJA1 disrupts the TGF-β tumor suppressor pathway, and its overexpression promotes cancer cell survival and proliferation. We have identified 2 novel triterpenoids that exhibit PRAJA1 inhibitory activity, which have demonstrated that suppression of PRAJA1 by specific small molecule inhibitors restores TGF-β tumor suppressor function and suppresses growth of HCC cells. Our results provide a better understanding of the mechanisms that regulate HCC development. Importantly, this study may lead to new therapeutics targeting this lethal cancer and potentially to a Phase I clinical trial in HCC. Citation Format: Jian Chen, Jiun-Sheng Chen, Vivek Shukla, Zhixing Yao, Hai-Long Piao, Wilma Jogunoori, Bibhuti Mishra, Lopa Mishtra. Targeting E3 ligase PRAJA1 in hepatocellular cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5198. doi:10.1158/1538-7445.AM2013-5198

Up-Regulation of Platelet-Activating Factor Synthases and its Receptor in Spinal Cord Contribute to Development of Neuropathic Pain following Peripheral Nerve Injury

BackgroundPlatelet-activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a lipid mediator derived from cell membrane. It has been reported that PAF is involved in various pathological conditions, such as spinal cord injury, multiple sclerosis, neuropathic pain and intrathecal administration of PAF leads to tactile allodynia. However, the expression of PAF synthases and its receptor in the spinal cord following peripheral nerve injury is unknown.MethodsUsing the rat spared nerve injury (SNI) model, we investigated the expression of PAF synthases (LPCAT1 and 2) and PAF receptor (PAFr) mRNAs in the spinal cord. Reverse transcription polymerase chain reaction (RT-PCR) and double-labeling analysis of in situ hybridization histochemistry (ISHH) with immunohistochemistry (IHC) were employed for the analyses. Pain behaviors were also examined with PAFr antagonist (WEB2086).ResultsRT-PCR showed that LPCAT2 mRNA was increased in the ipsilateral spinal cord after injury, but not LPCAT1 mRNA. Double-labeling of ISHH with IHC revealed that LPCAT1 and 2 mRNAs were constitutively expressed by a subset of neurons, and LPCAT2 mRNA was increased in spinal microglia after nerve injury. RT-PCR showed that PAFr mRNA was dramatically increased in the ipsilateral spinal cord after nerve injury. Double-labeling analysis of ISHH with IHC revealed that after injury PAFr mRNA was predominantly colocalized with microglia in the spinal cord. Continuous intrathecal administration of the PAFr antagonist suppressed mechanical allodynia following peripheral nerve injury. Delayed administration of a PAFr antagonist did not reverse the mechanical allodynia.ConclusionsOur data show the histological localization of PAF synthases and its receptor in the spinal cord following peripheral nerve injury, and suggest that PAF/PAFr signaling in the spinal cord acts in an autocrine or paracrine manner among the activated microglia and neurons, thus contributing to development of neuropathic pain.

Central and peripheral apelin receptor distribution in the mouse: Species differences with rat

The G protein-coupled apelin receptor (APJ) binds the endogenous peptide apelin and has been shown to have roles in many physiological systems. Thus far, distribution studies have predominantly been conducted in the rat and there is limited knowledge of the cellular distribution of APJ in mouse or human tissues. As recent functional studies have been conducted in APJ knock-out mice (APJ KO), in this study we undertook to characterize APJ mRNA and I125[Pyr1]apelin-13 binding site distribution in mouse tissues to enable correlation of distribution with function. We have utilized in situ hybridization histochemistry (ISHH) using APJ riboprobes, which revealed strong hybridization specifically in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus and in the anterior pituitary, with marginally lower levels in the posterior pituitary. In the periphery, strong hybridization was observed in the lung, heart, adrenal cortex, renal medulla, ovary and uterus. Autoradiographic binding to APJ with I125[Pyr1]apelin-13 exhibited significant binding in the anterior pituitary, while lower levels were observed in the posterior pituitary and PVN and SON. In the periphery, strong receptor binding was observed in tissues exhibiting intense riboprobe hybridization, indicating a good correlation between receptor transcription and translation. While the distribution of APJ mRNA and functional protein in the mouse shows similarities to that of the rat, we report a species difference in central APJ distribution and in the pituitary gland.

Sexually dimorphic expression of hypothalamic estrogen receptors α and β and Kiss1 in neonatal male and female rats.

Release of gonadotropins in adult rodents is sex specific and dependent upon kisspeptin (Kiss1) neurons. This crucial pathway within the hypothalamic-pituitary-gonadal (HPG) axis is profoundly influenced by neonatal estrogens, which induce a male-like phenotype. Classically, estrogen activity is mediated via the estrogen receptors α and β (ERα and ERβ), but the relative roles each plays in organizing the sex-specific ontogeny of kisspeptin signaling pathways remain unresolved. Thus, the present study used in situ hybridization histochemistry (ISHH) to map the temporal and sexually dimorphic neonatal mRNA expression profiles of ERα, ERβ, and Kiss1 in the anterioventral periventricular nucleus (AVPV), medial preoptic area (MPOA), ventromedial nucleus (VMN), and arcuate nucleus (ARC), all regions critical for kisspeptin regulation of gonadotropin secretion. In general, females had higher levels of ERα, in all regions examined, a sex difference that persisted until postnatal day (PND) 19 except in the ARC. Males had significantly more ERβ expression in the AVPV at birth, but this sex difference was lost and then re-emerged on PND 19, with females having more than males. VMN ERβ levels were higher in females until PND 19. Kiss1 was not detectable until PND 11 in the anterior hypothalamus, but expression levels were equivalent at birth in the ARC. By PND 2, ARC ERα and Kiss1 levels were abundant, sexually dimorphic (higher in females), and, respectively, showed a U- and a bell-shaped pattern with age. Sex differences in ARC Kiss1 expression provide evidence that Kiss1 may play a role in the sexual dimorphic organization of the neonatal brain. These detailed profiles of neonatal Kiss1 and ERs mRNA levels will help elucidate the relative roles each plays in the sex-specific, estrogen-dependent organization of gonadotropin signaling pathways.

Androgen receptor location in the dark-eyed junco using a probe for in situ hybridization histochemistry generated from zebra finch cDNA

Due to the role of sex steroids, namely testosterone (T), in the development and production of song in songbirds, androgen receptor (AR) densities in the brain regions controlling this behavior (i.e., the song control system) have long been studied. Many methods have been used to determine AR density and location to investigate the functional role of T in song development and production; however, a riboprobe developed from zebra finch (Taeniopygia guttata) cDNA was shown to be much more sensitive than previous methods. The zebra finch is a common model for song development and is sexually dimorphic, but does not breed seasonally or display seasonal changes in song control region volume. In this study, we used this riboprobe for in situ hybridization histochemistry (ISHH) to describe AR mRNA location in the brain of the dark-eyed junco (Junco hyemalis), a seasonally breeding model for which T has been shown to be important. Additionally, we provide a detailed comparison of AR mRNA location between these species. We found that this probe is indeed highly sensitive. We detected AR mRNA in four major regions of the song control system (HVC, MAN, RA and Area X). Additionally, we found that the location of AR mRNA in other regions varied only slightly between these two species. These findings suggest that this method is suitable for use across songbirds and it could be useful in the ongoing attempts to elucidate the roles of sex steroid hormones on the development of this and other sex steroid dependent behaviors in songbirds.

Heterogeneous distribution of the serotonin 5‐HT1A receptor mRNA in chemically identified neurons of the mouse rostral brainstem: Implications for the role of serotonin in the regulation of wakefulness and REM sleep

The 5-HT(1A) receptor (5-HT(1A)R) plays a key role in the inhibitory influence of serotonin (5-HT) on rapid eye movement (REM) sleep in rodents. However, the neuronal networks mediating such influence are mostly unknown, notably in the mouse. This led us to map 5-HT(1A)R mRNA, by in situ hybridization histochemistry (ISHH), and to characterize the neuronal phenotype of 5-HT(1A)R mRNA-positive neurons by dual ISHH and ISHH combined with immunohistochemistry, throughout the mouse rostral brainstem, a pivotal region for the generation of REM sleep and cortical activation. 5-HT(1A)R mRNA was found in most 5-HT neurons in the dorsal raphe (DR), the median raphe (MnR), the B9, and the interpeduncular (IP) nuclei. 5-HT(1A)R mRNA-positive neurons were also identified in individualized clusters of gamma-aminobutyric acid (GABA)ergic neurons in the DR and in neurons of an undetermined phenotype in the MnR. In addition, 1) GABAergic neurons of the ventral portion of Gudden's dorsal tegmental nucleus (DTg), the IP, and the caudal portion of the deep mesencephalic nucleus (DpMe), and 2) glutamatergic neurons scattered in the caudal pontine reticular nucleus (PnC) and densely packed in the internal lateral parabrachial subnucleus (PBil) also expressed 5-HT(1A)R mRNA. In contrast, no specific 5-HT(1A)R-related ISHH signal was generally detected in brainstem cholinergic and catecholaminergic neurons. These results emphasize the role of 5-HT(1A)R as an autoreceptor and the phenotypical heterogeneity of 5-HT(1A)R-expressing neurons within the DR and the MnR in the mouse brain. They also provide a neuroanatomical basis for understanding the influence of 5-HT(1A)R on REM sleep and wakefulness.

Expression of Cdc25B mRNA in Duodenal Mucosa of Chicken

Cdc25B is a mitotic regulator that might act as a starter phosphatase to initiate the positive feedback loop at the entry into mitotic (M) phase. In the present study, distribution of Cdc25B mRNA in duodenal mucosa of the chicken was demonstrated by means of in situ hybridization histochemistry (ISHH) using sense and antisense digoxigenin (DIG)-labeled RNA probes. The results showed that there were many labeled cells distributing in the duodenal mucosa of the adult chicken. Of these labeled cells, 81.60±9.63% of Cdc25B mRNA positive cells was distributed in the basilar part and mid-portion of the intestinal gland and 36.21±8.81% in the middle and basilar portion of villi of the small intestine of the chicken, respectively. Most of these labeled cells were positive in the regions of the stem cell and proliferation. The signals of ISHH decreased from basilar to upper part in the crypt of Lieberkuhn and weakened in the inferior villi of the duodenum. Moreover, the positive signals were both in the cytoplasm and cell nucleus. However, the labeled cells were negative in both the lamina muscularis mucosae and muscular layer. The results of ISHH suggested the existence of Cdc25B mRNA and vigorous proliferation activities in the duodenal mucosa of adult chicken, replenishing the cells which had sloughed off from the superior part of the villus. Our results provide some molecular evidence for a regular pattern of avian intestinal epitheliosis and functional partition and provide an approach to further study of the locations of Cdc25B in the chicken.

Expression of Leukotriene Receptors in the Rat Dorsal Root Ganglion and the Effects on Pain Behaviors

BackgroundLeukotrienes (LTs) belong to the large family of lipid mediators implicated in various inflammatory conditions such as asthma and rheumatoid arthritis. Four distinct types (BLT1, BLT2, CysLT1 and CysLT2) of G-protein-coupled receptors for LTs have been identified. Several studies have reported that LTs are involved in inflammatory pain, but the mechanism and the expression of LT receptors in the nociceptive pathway are unknown.ResultsWe investigated the precise expression of these four types of LT receptors in the adult rat dorsal root ganglion (DRG) using reverse transcription-polymerase reaction (RT-PCR) and radioisotope-labeled in situ hybridization histochemistry (ISHH). We detected mRNAs for BLT1 and CysLT2 in the DRG, but not for BLT2 and CysLT1. CysLT2 mRNA was preferentially expressed by small sized DRG neurons (about 36% of total neurons), whereas BLT1 mRNA was expressed by non-neuronal cells. Double labeling analysis of CysLT2 with NF-200, calcitonin gene-related peptide (CGRP), isolectin B4 (IB4), transient receptor potential vanilloid subfamily 1 (TRPV1) and P2X3 receptor revealed that many CysLT2-labeled neurons were localized with unmyelinated and non-peptidergic neurons, and interestingly, CysLT2 mRNA heavily co-localized with TRPV1 and P2X3-positive neurons. Intraplantar injection of LTC4, a CysLT2 receptor agonist, itself did not induce the thermal hyperalgesia, spontaneous pain behaviors or swelling of hind paw. However, pretreatment of LTC4 remarkably enhanced the painful behaviors produced by alpha, beta-methylene adenosine 5'-triphosphate (αβ-me-ATP), a P2X3 receptor agonist.ConclusionsThese data suggests that CysLT2 expressed in DRG neurons may play a role as a modulator of P2X3, and contribute to a potentiation of the neuronal activity following peripheral inflammation.

Nutritional state influences Nociceptin/Orphanin FQ peptide receptor expression in the dorsal raphe nucleus

Agonists of the nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor stimulate food intake. Concordantly, neuroanatomical localization of NOP receptor mRNA has revealed it to be highly expressed in brain regions associated with the regulation of energy balance. However, the specific mechanisms and neurochemical pathways through which physiological N/OFQ influences appetite are not well understood. To investigate this, we examined nutritional state-associated changes in NOP receptor mRNA levels throughout the rostrocaudal extent of the rat brain using in situ hybridization histochemistry (ISHH) and quantitative densitometry analysis. We observed a significant downregulation of NOP receptor mRNA in the dorsal raphe nucleus (DRN) of fasted rats compared to free-feeding rats. In contrast, no difference in NOP receptor mRNA expression was observed in the supraoptic, parventricular, ventromedial, arcuate or dorsomedial nuclei of the hypothalamus, the red nucleus, the locus coeruleus or the hypoglossal nucleus in the fasted or fed state. These data suggest that the endogenous N/OFQ system is responsive to changes in energy balance and that NOP receptors specifically within the DRN may be physiologically relevant to N/OFQ's effects on appetite.

Long-term survival of rats using self bone marrow cell engraftment in transplanted livers after liver transplantation

Objective To investigate the mechanisms of the long-term survival rats after using self bone marrow cell engraftment in transplanted livers liver transplantation.Methods The female rats receiving orthotopic liver transplantation were randomized into the blank control group (Group A), D hanks solution group (Group B), whole bone marrow cell group (Group C) and mesenchymal stem cell (MSCs) group (Group D).The blood levels of alanine transaminase (ALT) and total bili ru-bin (TBil) were determined 1 week after the transplantation.Liver pathological changes and survival time were also analyzed.The differentiation of the self bone marrow cells were assessed 60 days after transplantation using in situ hybridization histochemistry (ISHH)for Sry gene and alpha fetoprotein (AFP) or albumin immunohistochemistry(IHC).Results As compared with group A and B, MST of group C and D was significantly increased(P 0.05).As compared with group A and B, liver function 1 week after opera-tion was improved a lot in group C, D(P 0.05).The group A and B showed more serious acute rejection in 10 days after the operation, but there was little evidence of acute rejection in group C, D 60 days after the operation.ISHH for Sry gene and AFP or Albumin IHC double labeled cells were observed in group C and D.The percentage of the double staining positive ceils in group C was similar to that in group D(P>0.05).Conclusion Infusion of self bone marrow cells through the portal vein following liver transplantation could alleviate the acute rejection and induce long-term liver graft survival.MSCs can differentiate into hepatocytes expressing AFP or albumin.This may be the mechanism of the long-term liver graft survival. Key words: Liver transplantation; Rat; Self bone marrow cells; Mesenchymal stem cells; Acute rejection, Long-term survival

Taste disturbance after stapes surgery – clinical and experimental study

Conclusion: Most of the clinical cases experienced taste disturbance after stapes surgery, and in a few cases this disturbance persisted for a long time. The animal experiment suggested the role of geniculate ganglion (GG) cells in nerve generation. Objectives: To clinically examine taste disorder and its recovery after stapes surgery and experimentally demonstrate a role of GG. Patients and methods: Taste function after preservation of chorda tympani nerve (CTN) in stapes surgery was prospectively investigated with a questionnaire and electrogustometry (EGM). Further, expression of neurotrophic factors in GG after injury of CTN was examined by in situ hybridization histochemistry (ISSH) and RT-PCR. Results: Among the cases, 15/18 (83.3%) were associated with taste disturbance and 6/18 (33.3%) were associated with tongue numbness 2 weeks after surgery; however, the symptoms ceased in 14/18 cases (77.8%). Two weeks after surgery, the EGM threshold was found to be elevated in 15/18 cases (83.3%), while in 10/18 cases (55.6%), it did not decrease until 1 year after surgery. Expression of ISSH and amplified bands of BDNF and GFR increased at 7 and 14 days after nerve injury in ipsilateral GGs and also increased at 7 days on the contralateral side.

Circulating prolactin, MPOA prolactin receptor expression and maternal aggression in lactating rats

Maternal aggression is most intense in lactating rats from the 3rd to the 12th day postpartum. The purpose of this study was to determine if plasma prolactin (PRL) and prolactin receptor (PRL-R L) mRNA expression in the medial preoptic area (MPOA) of lactating rats are altered in association with maternal aggression. Lactating Sprague Dawley rats were divided into five groups and exposed for 10 min to an intruder male or to an object on postpartum day 8. Trunk blood and the brain of the dams were collected 30 or 240 min after exposure and from a non-exposed group. Lower levels of prolactin were found 30 min after the aggression test. No change was detected in the number of cells expressing PRL-R L mRNA by in situ hybridization histochemistry (ISHH) as a function of testing. However, the correlation between plasma PRL and PRL-R L mRNA expression in the mothers changed from positive in control females to negative in intruder exposed animals. These data support the concept that a maternal aggressive experience, while acutely altering PRL secretion, fails to affect PRL-R L mRNA expression.

Expression Profiles of Mouse Kell, XK, and XPLAC mRNA

Kell and XK are related because in red cells they exist as a disulfide-bonded complex. Kell is an endothelin-3-converting enzyme, and XK is predicted to be a transporter. Absence of XK, which is accompanied by reduced Kell on red cells, results in acanthocytosis and late-onset forms of central nervous system and neuromuscular abnormalities that characterize the McLeod syndrome. In this study, expression of mouse XK, XPLAC, a homolog of XK, and Kell were compared by in situ hybridization histochemistry (ISHH) and RT-PCR. ISHH showed that Kell and XK are coexpressed in erythroid tissues. ISHH detected XK, but not Kell, mRNA in testis, but RT-PCR indicated that both Kell and XK are coexpressed. XK, but not Kell, was significantly expressed in brain, spinal cord, small intestine, heart, stomach, bladder, and kidney. ISHH did not detect XK in skeletal muscle but RT-PCR did. In brain, XK was predominantly expressed in neuronal rather than in supportive cells. By contrast, XPLAC was predominantly expressed in the thymus. Coexpression of Kell and XK in erythroid tissues and the different expressions in non-erythroid tissues suggest that XK may have a complementary hematological function with Kell and a separate role in other tissues.

Effect of Fasting on Cocaine-Amphetamine-Regulated Transcript, Neuropeptide Y, and Leptin Receptor Expression in the Non-Human Primate Hypothalamus

Leptin is a cytokine produced by white adipose tissue that circulates in direct proportion to adiposity and is an important signal of energy balance. Leptin inhibits food intake in rodents by inhibiting the orexigenic neuropetides neuropeptide Y (NPY) and agouti regulated peptide (AgRP) and stimulating the anorexigenic neuropeptides α-melanocyte-stimulating hormone (α-MSH) and cocaine-amphetamine-regulated transcript (CART). In order to extend our understanding of neuroendocrine regulation of appetite in the primate, we determined the effect of a metabolic challenge on CART, NPY, and leptin receptor (Ob-R) messenger ribonucleic acid (mRNA) in the nonhuman primate (NHP) hypothalamus. Ten adult female rhesus monkeys were either maintained on a regular diet or fasted for two days before euthanasia. CART, NPY, and Ob-R mRNA were measured by in situ hybridization histochemistry (ISHH). A 2-day fast decreased CART expression in the ARC, increased NPY gene expression in the supraoptic nucleus (SON) and paraventricular nucleus (PVN), and increased Ob-R expression in the ventromedial nucleus (VMN). This is the first report that fasting inhibits CART expression and stimulates Ob-R expression in monkeys. Increased NPY expression in the SON and PVN, but not the ARC of fasted monkeys also is novel. With some exceptions, our observations are confirmatory of findings in rodent studies. Similarities in the neuroendocrine responses to a metabolic challenge in monkeys and rodents support extending existing hypotheses of neuroendocrine control of energy homeostasis to primates.

The vasopressin 1b receptor is prominent in the hippocampal area CA2 where it is unaffected by restraint stress or adrenalectomy

The vasopressin 1b receptor (Avpr1b) is one of two principal receptors mediating the behavioral effects of vasopressin (Avp) in the brain. Avpr1b has recently been shown to strongly influence social forms of aggression in mice and hamsters. This receptor appears to play a role in social recognition and motivation as well as in regulating the hypothalamic–pituitary–adrenal axis. Most of these studies have been performed in knockout mice, a species in which the localization of the Avpr1b has not been described, thus precluding correlations with the behaviors. We performed in situ hybridization histochemistry (ISHH) with specific probes and found especially prominent expression within the CA2 pyramidal neurons of the hippocampus, with much lower expression in the hypothalamic paraventricular nucleus and amygdala. Reverse transcriptase–polymerase chain reaction (RT-PCR) confirmed expression in those as well other areas in which the ISHH was not sensitive enough to detect labeled cells (e.g. piriform cortex, septum, caudate-putamen and lower brainstem areas). Mouse Avpr1b transcript levels were not altered in the CA2 field by restraint stress or adrenalectomy. Finally, ISHH and RT-PCR showed expression of the Avpr1b gene in the rat and human hippocampi as well. We suggest that the CA2 field may form or retrieve associations (memories) between olfactory cues and social encounters.