Preconditioning-Induced Facilitation of Lactate Release from Astrocytes Is Essential for Brain Ischemic Tolerance.

A sublethal ischemic episode [termed preconditioning (PC)] protects neurons in the brain against a subsequent severe ischemic injury. This phenomenon is known as brain ischemic tolerance and has received much attention from researchers because of its robust neuroprotective effects. We have previously reported that PC activates astrocytes and subsequently upregulates P2X7 receptors, thereby leading to ischemic tolerance. However, the downstream signals of P2X7 receptors that are responsible for PC-induced ischemic tolerance remain unknown. Here, we show that PC-induced P2X7 receptor-mediated lactate release from astrocytes has an indispensable role in this event. Using a transient focal cerebral ischemia model caused by middle cerebral artery occlusion, extracellular lactate levels during severe ischemia were significantly increased in mice who experienced PC; this increase was dependent on P2X7 receptors. In addition, the intracerebroventricular injection of lactate protected against cerebral ischemic injury. In in vitro experiments, although stimulation of astrocytes with the P2X7 receptor agonist BzATP had no effect on the protein levels of monocarboxylate transporter (MCT) 1 and MCT4 (which are responsible for lactate release from astrocytes), BzATP induced the plasma membrane translocation of these MCTs via their chaperone CD147. Importantly, CD147 was increased in activated astrocytes after PC, and CD147-blocking antibody abolished the PC-induced facilitation of astrocytic lactate release and ischemic tolerance. Taken together, our findings suggest that astrocytes induce ischemic tolerance via P2X7 receptor-mediated lactate release.

The LAT1 inhibitor JPH203 suppresses the growth of castration-resistant prostate cancer through a CD24-mediated mechanism.

L-type amino acid transporter 1 (LAT1) is specifically expressed in many malignancies, contributes to the transport of essential amino acids, such as leucine, and regulates the mammalian target of rapamycin (mTOR) signaling pathway. We investigated the expression profile and functional role of LAT1 in prostate cancer using JPH203, a specific inhibitor of LAT1. LAT1 was highly expressed in castration-resistant prostate cancer (CRPC) cells, including C4-2 and PC-3 cells, but its expression level was low in castration-sensitive LNCaP cells. JPH203 significantly inhibited [14C] leucine uptake in CRPC cells but had no effect in LNCaP cells. JPH203 inhibited the proliferation, migration, and invasion of CRPC cells but not of LNCaP cells. In C4-2 cells, Cluster of differentiation (CD) 24 was identified by RNA sequencing as a novel downstream target of JPH203. CD24 was downregulated in a JPH203 concentration-dependent manner and suppressed activation of the Wnt/ß-catenin signaling pathway. Furthermore, an in vivo study showed that JPH203 inhibited the proliferation of C4-2 cells in a castration environment. The results of this study indicate that JPH203 may exert its antitumor effect in CRPC cells via mTOR and CD24.

Central administered xenin induced Fos expression in nesfatin-1 neurons in rats.

Xenin is a 25-amino acid peptide identified in human gastric mucosa, which is widely expressed in peripheral and central tissues. It is known that the central or peripheral administration of xenin decreases food intake in rodents. Nesfatin-1/NUCB2 (nesfatin-1) has been identified as an anorexic neuropeptide, it is often found co-localized with many peptides in the central nervous system. After the intracerebroventricular administration of xenin on nesfain-1-like immunoreactivity (LI) neurons, we examined its effects on food intake and water intake in rats. As a result, Fos-LI neurons were observed in the organum vasculosum of the laminae terminalis (OVLT), the median preoptic nucleus (MnPO), the subfornical organ (SFO), the supraoptic nucleus (SON), the paraventricular nucleus (PVN), the arcuate nucleus (Arc), the lateral hypothalamic area (LHA), the central amygdaloid nucleus (CAN), the dorsal raphe nucleus (DR), the locus coeruleus (LC), the area postrema (AP) and the nucleus of the solitary tract (NTS). After the administration, the number of Fos-LI neurons was significantly increased in the LC and the OVLT, the MnPO, the SFO, the SON, the PVN, the Arc, the LHA, the CAN, the DR, the AP and the NTS, compared with the control group. After the administration of xenin, we conducted double immunohistochemistry for Fos and nesfatin-1, and found that the number of nesfatin-1-LI neurons expressing Fos were significantly increased in the SON, the PVN, the Arc, the LHA, the CAN, the DR, the AP and the NTS, compared with the control group. The pretreatment of nesfatin-1 antisense significantly attenuated this xenin-induced feeding suppression, while that of nesfatin-1 missense showed no improvement. These results indicate that central administered xenin may have anorexia effects associated with activated central nesfatin-1 neurons.

Determinants of quality of life in degenerative cervical myelopathy: a systematic review.

BACKGROUND: Degenerative cervical myelopathy (DCM) is the most common cause of chronic, progressive spinal cord impairment worldwide. Patients experience substantial pain, functional neurological decline and disability. Health-related quality of life (HRQoL) appears to be particularly poor, even when compared to other chronic diseases. However, the determinants of HRQoL are poorly understood. The objective was to perform a systematic review of the determinants of quality of life of people with DCM. METHODS: A systematic search was conducted in MEDLINE and Embase following PRISMA 2020 guidelines (PROSPERO CRD42018115675). Full-text papers in English, exclusively studying DCM, published before 26 March 2020 were eligible for inclusion and were assessed using the Newcastle-Ottawa Scale and the Cochrane Risk of Bias 2 (RoB 2) tool. Study sample characteristics, patient demographics, cohort type, HRQoL instrument utilised, HRQoL score, and relationships of HRQoL with other variables were qualitatively synthesised. RESULTS: A total of 1176 papers were identified; 77 papers and 13,572 patients were included in the final analysis. A total of 96% of papers studied surgical cohorts and 86% utilised the 36-Item Short Form Survey (SF-36) as a measure of HRQoL. HRQoL determinants were grouped into nine themes. The most common determinant to be assessed was surgical technique (38/77, 49%) and patient satisfaction and experience of pain (10/77, 13%). HRQoL appeared to improve after surgery. Pain was a negative predictor of HRQoL. CONCLUSION: Current data on the determinants of HRQoL in DCM are limited, contradictory and heterogeneous. Limitations of this systematic review include lack of distinction between DCM subtypes and heterogenous findings amongst the papers in which HRQoL is measured postoperatively or post-diagnosis. This highlights the need for greater standardisation in DCM research to allow further synthesis. Studies of greater precision are necessary to account for HRQoL being complex, multi-factorial and both time and context dependent.

Effects of a novel hepatitis B anti-viral drug E-CFCP in renal organic acid transporters.

Currently, the emergence of drug resistance is an important issue in the treatment of hepatitis B virus (HBV). Recently, our collaborating group developed a novel long-acting anti-HBV drug, E-CFCP. However, until this study, the effects of E-CFCP in the kidney have remained unclarified. Using cell viability and uptake assays, we examined the effects of E-CFCP on the function of renal organic anion transporters (OATs). No cytotoxicity was shown related to the E-CFCP in the renal OATs in either assay. Thus, this study suggested that E-CFCP may be a novel, excellent candidate drug for the treatment of drug-resistant HBV.

Targeting L-type amino acid transporter 1 in urological malignancy: Current status and future perspective.

Amino acid transporters are responsible for the uptake of amino acids, critical for cell proliferation. L-type amino acid transporters play a major role in the uptake of essential amino acids. L-type amino acid transporter 1 (LAT1) exerts its functional properties by forming a dimer with 4F2hc. Utilizing this cancer-specificity, research on diagnostic imaging and therapeutic agents for malignant tumors targeting LAT1 progresses in various fields. In hormone-sensitive prostate cancer, the up-regulation of L-type amino acid transporter 3 (LAT3) through the androgen receptor (AR) has been identified. On the other hand, in castration-resistant prostate cancer, the negative regulation of LAT1 through AR has been determined. Furthermore, 4F2hc: a binding partner of LAT1, was identified as the specific downstream target of Androgen Receptor Splice Variant 7: AR-V7. LAT1 has been suggested to contribute to acquiring castration resistance in prostate cancer, making LAT1 a completely different therapeutic target from anti-androgens and taxanes. Increased expression of LAT1 has also been found in renal and bladder cancers, suggesting a contribution to acquiring malignancy and progression. In Japan, clinical trials of LAT1 inhibitors for solid tumors are in progress, and clinical applications are now underway. This article will summarize the relationship between LAT1 and urological malignancies.

Ischemic Tolerance Induced by Glial Cells.

Ischemic tolerance is a phenomenon in which resistance to subsequent invasive ischemia is acquired by a preceding noninvasive ischemic application, and is observed in many organs, including the brain, the organ most vulnerable to ischemic insult. To date, much research has been conducted on cerebral ischemic tolerance as a cell-autonomous action of neurons. In this article, we review the essential roles of microglia and astrocytes in the acquisition of ischemic tolerance through neuron-non-autonomous mechanisms, where the two types of glial cells function in a concerted manner to induce ischemic tolerance.

P2X7 Receptors in Astrocytes: A Switch for Ischemic Tolerance.

A sub-lethal ischemic episode (preconditioning [PC]) protects neurons against a subsequent lethal ischemic injury. This phenomenon is known as ischemic tolerance. PC itself does not cause brain damage, but affects glial responses, especially astrocytes, and transforms them into an ischemia-resistant phenotype. P2X7 receptors (P2X7Rs) in astrocytes play essential roles in PC. Although P2X7Rs trigger inflammatory and toxic responses, PC-induced P2X7Rs in astrocytes function as a switch to protect the brain against ischemia. In this review, we focus on P2X7Rs and summarize recent developments on how astrocytes control P2X7Rs and what molecular mechanisms they use to induce ischemic tolerance.

Tremor as a symptom of degenerative cervical myelopathy: a systematic review.

BACKGROUND: AO Spine RECODE-DCM (Research objectives and common data elements for degenerative cervical myelopathy) has highlighted that the subjective disability reported by people living with DCM is much broader than routinely considered today by most professionals. This includes a description of tremor. The objective of this review was to study the incidence and possible aetiology of tremor in degenerative cervical myelopathy (DCM). METHODS: A systematic review registered in PROSPERO (CRD42020176905) was conducted in Embase and MEDLINE for papers studying tremor and DCM published on or before the 20th of July 2020. All manuscripts describing an association between tremor and DCM in humans were included. Articles relating to non-human animals, and those not available in English were excluded. An analysis was conducted in accordance with PRISMA and SWiM guidelines for systematic reviews. RESULTS: Out of a total of 4402 screened abstracts, we identified 7 case reports and series describing tremor in 9 DCM patients. Papers were divided into three groups for the discussion. The first group includes DCM correctly identified on presentation, with tremor as a described symptom. The second group includes cases where DCM was misdiagnosed, often as Parkinson's disease. The third group includes a single case with a previous history of DCM, presenting with an otherwise unexplained tremor. This grouping allows for the clustering of cases supporting various arguments for the association between tremor and DCM. CONCLUSION: DCM can be associated with tremor. The current evidence is restricted to case series. Further study is warranted to establish tremor prevalence, and its significance to assessment and management.

Gathering Global Perspectives to Establish the Research Priorities and Minimum Data Sets for Degenerative Cervical Myelopathy: Sampling Strategy of the First Round Consensus Surveys of AO Spine RECODE-DCM.

STUDY DESIGN: Survey. INTRODUCTION: AO Spine Research Objectives and Common Data Elements for Degenerative Cervical Myelopathy (AO Spine RECODE-DCM) is an international initiative that aims to accelerate knowledge discovery and improve outcomes by developing a consensus framework for research. This includes defining the top research priorities, an index term and a minimum data set (core outcome set and core data elements set - core outcome set (COS)/core data elements (CDE)). OBJECTIVE: To describe how perspectives were gathered and report the detailed sampling characteristics. METHODS: A two-stage, electronic survey was used to gather and seek initial consensus. Perspectives were sought from spinal surgeons, other healthcare professionals and people with degenerative cervical myelopathy (DCM). Participants were allocated to one of two parallel streams: (1) priority setting or (2) minimum dataset. An email campaign was developed to advertise the survey to relevant global stakeholder individuals and organisations. People with DCM were recruited using the international DCM charity Myelopathy.org and its social media channels. A network of global partners was recruited to act as project ambassadors. Data from Google Analytics, MailChimp and Calibrum helped optimise survey dissemination. RESULTS: Survey engagement was high amongst the three stakeholder groups: 208 people with DCM, 389 spinal surgeons and 157 other healthcare professionals. Individuals from 76 different countries participated; the United States, United Kingdom and Canada were the most common countries of participants. CONCLUSION: AO Spine RECODE-DCM recruited a diverse and sufficient number of participants for an international PSP and COS/CDE process. Whilst PSP and COS/CDE have been undertaken in other fields, to our knowledge, this is the first time they have been combined in one process.

High Prevalence of Pre-Existing Liver Abnormalities Identified Via Autopsies in COVID-19: Identification of a New Silent Risk Factor?

A high prevalence of hepatic pathology (in 17 of 19 cases) was reported in post-mortem (PM) examinations of COVID-19 patients, undertaken between March 2020 and February 2021 by a single autopsy pathologist in two English Coronial jurisdictions. The patients in our cohort demonstrated high levels of recognised COVID-19 risk factors, including hypertension (8/16, 50%), type 2 diabetes mellitus (8/16, 50%) and evidence of arteriopathy 6/16 (38%). Hepatic abnormalities included steatosis (12/19; 63%), moderate to severe venous congestion (5/19; 26%) and cirrhosis (4/19; 21%). A subsequent literature review indicated a significantly increased prevalence of steatosis (49%), venous congestion (34%) and cirrhosis (9.3%) in COVID-19 PM cases, compared with a pre-pandemic PM cohort (33%, 16%, and 2.6%, respectively), likely reflecting an increased mortality risk in SARS-CoV-2 infection for patients with pre-existing liver disease. To corroborate this observation, we retrospectively analysed the admission liver function test (LFT) results of 276 consecutive, anonymised COVID-19 hospital patients in our centre, for whom outcome data were available. Of these patients, 236 (85.5%) had significantly reduced albumin levels at the time of admission to hospital, which was likely indicative of pre-existing chronic liver or renal disease. There was a strong correlation between patient outcome (length of hospital admission or death) and abnormal albumin at the time of hospital admission (p = 0.000012). We discuss potential mechanisms by which our observations of hepatic dysfunction are linked to a risk of COVID-19 mortality, speculating on the importance of recently identified anti-interferon antibodies.

Adenosine A2B receptor down-regulates metabotropic glutamate receptor 5 in astrocytes during postnatal development.

Metabotropic glutamate receptor 5 (mGluR5) in astrocytes is a key molecule for controlling synapse remodeling. Although mGluR5 is abundant in neonatal astrocytes, its level is gradually down-regulated during development and is almost absent in the adult. However, in several pathological conditions, mGluR5 re-emerges in adult astrocytes and contributes to disease pathogenesis by forming uncontrolled synapses. Thus, controlling mGluR5 expression in astrocyte is critical for several diseases, but the mechanism that regulates mGluR5 expression remains unknown. Here, we show that adenosine triphosphate (ATP)/adenosine-mediated signals down-regulate mGluR5 in astrocytes. First, in situ Ca2+ imaging of astrocytes in acute cerebral slices from post-natal day (P)7-P28 mice showed that Ca2+ responses evoked by (S)-3,5-dihydroxyphenylglycine (DHPG), a mGluR5 agonist, decreased during development, whereas those evoked by ATP or its metabolite, adenosine, increased. Second, ATP and adenosine suppressed expression of the mGluR5 gene, Grm5, in cultured astrocytes. Third, the decrease in the DHPG-evoked Ca2+ responses was associated with down-regulation of Grm5. Interestingly, among several adenosine (P1) receptor and ATP (P2) receptor genes, only the adenosine A2B receptor gene, Adora2b, was up-regulated in the course of development. Indeed, we observed that down-regulation of Grm5 was suppressed in Adora2b knockout astrocytes at P14 and in situ Ca2+ imaging from Adora2b knockout mice indicated that the A2B receptor inhibits mGluR5 expression in astrocytes. Furthermore, deletion of A2B receptor increased the number of excitatory synapse in developmental stage. Taken together, the A2B receptor is critical for down-regulation of mGluR5 in astrocytes, which would contribute to terminate excess synaptogenesis during development.

Mechanisms underlying sensitization of P2X7 receptors in astrocytes for induction of ischemic tolerance.

We previously showed that noninvasive mild ischemia (preconditioning; PC) induced ischemic tolerance by upregulation of P2X7 receptors in astrocytes via a hypoxia inducible factor-1α (HIF-1α)-dependent mechanism. The P2X7 receptor is known as a low-sensitivity P2 receptor that requires a high extracellular ATP (eATP) concentration for activation. PC increased the eATP level but was not sufficient to activate P2X7 receptors. Here, we show that astrocytes possess an elaborate mechanism for activation of P2X7 receptors, thus contributing to ischemic tolerance. Nicotinamide adenine dinucleotide (NAD+ ) was shown to increase the sensitivity of P2X7 receptors to eATP via ecto-ADP-ribosyltransferase 2 (ARTC2)-catalyzed ADP-ribosylation in peripheral immune cells. Although ARTC2-positive signals were mostly absent in the naïve brain, they were selectively increased in astrocytes by PC. The spatiotemporal pattern of PC-evoked ARTC2 was well associated with that of P2X7 receptors. In the in vitro experiments, NAD+ increased the sensitivity of P2X7 receptors to ATP, and at higher concentrations, NAD+ itself activated P2X7 receptors without eATP in cultured astrocytes. In the in vivo experiments using middle cerebral artery occlusion model mice, the PC-evoked increase in HIF-1α in astrocytes was abolished by the ARTC2 inhibitor S + 16a. S + 16a also abolished PC-evoked ischemic tolerance. Taken together, the results suggested that P2X7 receptors can be sensitized to ATP by NAD+ /ARTC2-catalyzed ADP-ribosylation, which allows astrocytes to drive P2X7 receptor-mediated ischemic tolerance even though PC only slightly increases the amount of eATP.

Effects of islatravir (4'-ethynyl-2-fluoro-2'-deoxyadenosine or EFdA) on renal tubular cells and islatravir's interactions with organic anion transporters.

Islatravir (ISL; 4'-ethynyl-2-fluoro-2'-deoxyadenosine or EFdA) is a novel reverse transcriptase translocation inhibitor and has a unique structure and high antiviral activity against wild-type and multidrug resistant HIV strains. In this study, we investigated whether islatravir (ISL) can cause kidney damage compared to tenofovir disoproxil fumarate (TDF) and tenofovir (TFV). We also investigated interactions of these drugs with organic anion transporters (OATs). There is a large gap in ISL concentration between the pharmacological dose to proximal tubular cells and the clinical dose. ISL is unlikely to be taken up via OAT1 or OAT3; therefore, OAT1 and OAT3 may not be involved in the injury to tubular cells. Present data strongly suggests that ISL is not toxic to proximal tubules because blood levels of ISL are not high enough to cause kidney damage in the clinical setting.

Protein kinase C activation upregulates human L-type amino acid transporter 2 function.

L-type amino acid transporter 2 (LAT2) is a Na+-independent neutral amino acid transporter, whose function regulation system remains unclarified. Since protein kinase C (PKC) is known to regulate the functions of various transporters, we investigated whether human LAT2 (hLAT2) function is regulated by PKC. In mouse proximal tubule S2 cells, hLAT2 transport activity was upregulated by PKC activation. However, we found that the mRNA and protein expression of hLAT2 was not affected by PKC activation and that the upregulation was independent of the three potential PKC consensus sites in the hLAT2 amino acid sequence. Moreover, we found that PKC activation upregulated the Vmax value for hLAT2-mediated alanine transport, which was not accompanied by the induction of hLAT2 membrane insertion. In conclusion, we showed that hLAT2 function is upregulated by PKC activation, which is not related to either the de novo synthesis, the phosphorylation or the membrane insertion of hLAT2.

Induction of short-term pseudopregnancy in gilts by the administration of estradiol benzoate or estradiol dipropionate to achieve ovulatory synchronization for embryo collection.

A study was conducted to investigate whether ovulation in gilts could be synchronized for embryo collection by the administration of estradiol benzoate (EB) or estradiol dipropionate (EDP) to induce pseudopregnancy, followed by the treatment with prostaglandin F2α (PGF2α ) on 10 days after. Ten gilts each received a total of 20 mg of EB or EDP on Day 10 or EB on Day 10 and 14 to induce pseudopregnancy (Day 0 = onset of estrus). Donors received PGF2α 10 or 15 days (as a control) after the first administration of estrogens and subsequently eCG and hCG, and were then inseminated artificially. The embryos were collected 7 days after the administration of hCG, and assessed for embryo yield and their developmental stages. All protocols resulted in good embryo yield (9.8-13.2 embryos in average), and the embryos showed average ability to develop to the expanded blastocyst stage (3.29-4.03 as developmental scores) without any significant differences among the protocols. These results suggest that the administration of PGF2α 10 days after the treatment of gilts with EB or EDP would allow synchronization of ovulation and embryo collection, as well as shortening the period from estrus detection to embryo collection, thus improving embryo collection efficiency.

Piglet production by non-surgical transfer of vitrified embryos, transported to commercial swine farms and warmed on site.

We investigated the feasibility of piglet production by non-surgical embryo transfer (Ns-ET) of vitrified porcine blastocysts and expanded blastocysts transported to commercial farms and warmed on site (V/T/W embryos). Ns-ET was performed by depositing 11-20 vitrified and warmed embryos at a proximal site within the uterus via a catheter. In Experiment 1, the effect of donor-recipient estrous cycle asynchrony on the efficiency of Ns-ET of vitrified and ordinary warmed embryos was investigated at the experimental facility. With a 1-day delay recipients relative to that of donor, the farrowing rate was 50.0% and the survival rate to term was 21.1%. In Experiment 2, Ns-ET using recipients with a 1-day delay and vitrified embryos after one-step warming and dilution was evaluated at the experimental facility. Although the resulting farrowing rate was 42.9%, the survival rate was 6.4%. In Experiment 3, Ns-ET was conducted using V/T/W embryos at four commercial farms, where piglets derived from them were produced. When artificial insemination was conducted prior to Ns-ET, the farrowing and survival rates obtained using V/T/W embryos were 75.0%, and 21.3%, respectively. These results show that Ns-ET of V/T/W embryos using this protocol would be feasible for piglet production at farms.

Non-surgical transfer of vitrified porcine embryos using a catheter designed for a proximal site of the uterus.

This study aimed to compare the efficiency of non-surgical embryo transfer (ET) using a newly developed catheter, which enables transferring embryos into a proximal site of the uterus (mostly uterine body), and surgical ET of vitrified porcine embryos. In Experiment 1, the catheter was inserted into 12 gilts, with each half of the group allocated to skilled or novice operators. The time required for insertion into the uterus did not differ between skilled and novice operators (4 min 9 s and 4 min 6 s, respectively). In Experiment 2, 12 gilts were used as recipients for non-surgical and surgical ET with vitrified embryos (n = 6, each). There was no significant difference in the rate of piglet production based on the number of transferred embryos between surgical and non-surgical ET (25.8% vs. 15.4%, p = .098). The results suggest that non-surgical ET catheter allowed for easy insertion and transfer of embryos without special training. Although the catheter is effective for deposition of embryos into the proximal site of uterus, the efficiency of piglet production is not enhanced compared with surgical ET. The ET method using this catheter, being labor-saving and less-invasive, may contribute to the improvement of ET in pigs.

Embryo collection from pigs post-pseudopregnancy induced by estradiol dipropionate.

We aimed to define whether embryo collection carried out after pseudopregnancy was of similar outcome and quality as after artificial abortion. To induce pseudopregnancy, 30 gilts or sows were given 20 mg intramuscular estradiol dipropionate (EDP) 10-11 days after the onset of estrus. Ten additional pigs were inseminated artificially at natural estrus as a control group. Prostaglandin F2α (PGF2α ) was administered twice with a 24 hr interval beginning 15, 20, or 25 days after EDP-treatment (n = 10 per group) or between 23 and 39 days after artificial insemination in control pigs. Following this, all pigs were given 1,000 IU equine chorionic gonadotropin and 500 IU human chorionic gonadotropin (hCG) and then inseminated. Embryos were recovered 6 or 7 days after hCG treatment and outcome was recorded. There was no significant difference in the number of normal embryos collected from the pigs with PGF2α initiated at different time points or from the control group. Embryonic developmental stages 7 days after hCG treatment also did not differ among groups. These results indicate that the use of EDP to induce pseudopregnancy, followed by PGF2α administration to synchronize estrus for subsequent embryo harvest, is a suitable alternative to the artificial abortion method.