Background: Antibiotic use prior to biopsy acquisition in people with diabetes and osteomyelitis of the foot (DFO) might influence bacterial yield in cultures or induce bacterial resistance. Obtaining reliable culture results is pivotal to guide antibiotics for conservative treatment of DFO. Methods: We prospectively analysed cultures of ulcer bed and percutaneous bone biopsies of people with DFO and investigated if antibiotics administered prior to (<2 months up to 7 days) biopsy acquisition led to more negative cultures or increased resistance in virulent bacteria. We calculated relative risks (RR) and 95% confidence intervals (CIs). We stratified analyses according to biopsy type (ulcer bed or bone). Results: We analysed bone and ulcer bed biopsies of 64 people, of whom 29 received prior antibiotics, and found that prior antibiotics did not lead to a higher risk of at least one negative culture , nor did prior treatment increase the risk of a specific type of negative culture , RR for ulcer bed cultures 0.92 (CI 0.33–2.6)) or both cultures (RR 1.3 (CI 0.35–4.7), and neither did it increase the risk of antibiotic resistance in the combined bacterial results of ulcer bed and bone cultures ). Conclusions: Antibiotics administered up to 7 days before obtaining biopsies in people with DFO do not influence culture yield regardless of biopsy type, nor are they associated with more antibiotic resistance. The cornerstone of conservative treatment of diabetic foot osteomyelitis (DFO) is antibiotic therapy based on susceptibility testing of cultures of an adequately obtained ulcer bed and/or bone biopsy ,2. SinceDFO usually starts with a soft tissue or ulcer infection spreading contiguously to the bone . A numbeAt present, it is unclear if antibiotic use prior to biopsy taking for DFO influences culture results or how long an antibiotic-free period should be before reliable biopsies can be obtained. We hypothesised that the administration of antibiotics within two months and up to 7 days prior to obtaining biopsies will lead to more negative cultures and a higher risk of antibiotic resistance in these cultures. To investigate this, we compared the culture results of people with prior antibiotic treatment with those without prior antibiotic treatment with regard to culture yield and antibiotic resistance.We included 64 people in this observational study. Of the 64 participants, 36 (56.3%) had at least one negative culture, and in 28 (43.8%), both cultures showed bacterial growth. Of the participants with prior antibiotic treatment, 62.0% had at least one specimen without growth versus 50.0% of participants without prior antibiotic treatment .When calculating relative risks with 95% confidence intervals, prior antibiotic use did not lead to a higher risk of at least one negative culture, nor did prior treatment increase the risk of sterility in a specific biopsy type (bone or ulcer bed) or both types . We inveWhen adding the number of antibiotic courses as a categorical variable to the model, the group of people with two prior antibiotic courses was too small, therefore the odds could not be estimated.In Staphylococcus aureus and Staphylococcus lugdunensis, beta-haemolytic streptococci, Enterobacterales, or Pseudomonas spp.) in either one of the biopsies. In 14 (29.8%) of these 47 participants, we identified at least one resistant species.Of the 64 participants included, 47 (73.3%) had growth of a virulent bacterial species /Enterococcus spp., and anaerobes more frequently  versus 89 bacterial species in 35 participants without prior treatment (average of 2.5 unique bacteria per person). We did not identify a lower number of bacteria in participants without prior treatment due to bacterial selection.S aureus, Enterococcus species, and Enterobacteriaceae. That study probably obtained different results compared to our study because intravenous antibiotics were administered in the 7 days prior to biopsy, in contrast with our participants who received oral antibiotics up to 7 days prior to biopsy and did not receive antibiotics during the 7 days directly preceding the biopsy. When comparing bacterial species, Young et al. identified more Streptococcus spp. and anaerobes in people with fewer hours of total antibiotic exposure, and we identified more Streptococcus (non-beta-haemolytic and beta-haemolytic taken together) and anaerobes in participants without prior antibiotics. Different to Young et al., we did identify more S. aureus in people without prior antibiotics, whereas Young et al. did not. Two abstracts reported contradictory results regarding the influence of antibiotic exposure prior to bone biopsy on culture yield in people with osteomyelitis [We examined the impact of previous antibiotic use on the bacterial yield and resistance in ulcer bed and bone biopsy cultures from persons with diabetes and foot osteomyelitis. We found that administering antibiotics up to 7 days before obtaining biopsies did not affect culture yield or increase the risk of antibiotic resistance, regardless of the type of culture. In the literature, there is controversy regarding the influence of prior antibiotics on culture yield. Young et al. retrospectively investigated the effect of pre-operative antibiotics on operatively-obtained soft tissue or bone specimens of people with diabetic foot infection . They fomyelitis ,13. One myelitis . The othmyelitis ,13.Besides some literature regarding lower extremity osteomyelitis, there are also some studies in vertebral osteomyelitis showing contradictory results ,14,15,16A strength of our study is that we investigated the influence of prior treatment on ulcer bed and bone cultures separately and combined. By doing this, we investigated several clinical scenarios . Bone biopsy is not common practice in all hospitals for the diagnosis and identification of pathogens in DFO. Ulcer bed biopsies are also obtained in case of soft tissue infection without osteomyelitis. Investigating the yield of bone and ulcer bed biopsies separately makes the results applicable for both soft tissue and bone infection. Additionally, by comparing both sampling methods, we eliminated the inequality of the pre-test likelihood of positive ulcer bed and bone cultures. Ulcer bed cultures are open to surroundings, which increases the pre-test likelihood that cultures of the ulcer bed become positive compared with bone. We did not find an increased risk for negative cultures in participants with prior antibiotic use with relatively small 95% confidence intervals, suggesting a low uncertainty of this finding. When we stratified participants according to the antibiotic-free period (exactly 7 days or longer than 7 days), groups became small with concomitant wider 95% confidence intervals, indicating a larger uncertainty of the obtained results, and one of the stratified groups even became too small to reliably estimate results with logistic regression analyses. Based on our results, an interval of at least 7 days might be safe, but larger randomised controlled trials should confirm these findings, and these further studies could investigate if an even shorter interval might also be feasible.In the present study, we did not find an association between prior antibiotics and the occurrence of resistant bacteria in either bone or ulcer bed cultures, although we investigated resistance according to a predefined clear definition based on guidelines and antimicrobial stewardships for the treatment of DFO ,18,19,20Our results showed a different relation between antibiotic exposure and resistance for participants with an antibiotic-free period longer than 7 days compared to participants with an antibiotic-free period of exactly 7 days in comparison to participants without prior antibiotic treatment. Based on the pathophysiological mechanisms of resistance induction and the literature, we do not believe that prior antibiotics can have a protective effect on resistance induction ,22,23,24We tried to investigate the influence of the number of antibiotic courses on the occurrence of resistance because we assumed that this variable could be of influence ,13,25. TCorynebacterium spp.). These unclassified results were, therefore, not taken into account in the analyses, and we had to accept some loss of power for this part of the study. This is reflected by the larger 95% confidence intervals in the logistic regression analyses.Because of our clear and unambiguous definition of resistance, we could not classify all culture results, i.e., cultures only consisting of uncommon gram-positive or -negative bacteria, anaerobes, or bacteria, which are usually intrinsically resistant ;○A higher risk of antibiotic resistance in cultures, with a focus on virulent bacteria. We evaluated if antibiotic use within 2 months and up to 7 days prior to biopsy lead to:We hypothesised that the administration of antibiotics shortly before obtaining biopsies will lead to more negative cultures, and that prior antibiotics lead to a higher risk of antibiotic resistance in virulent bacteria in these cultures.This is a prospective observational study, using data from cultures taken during the international multicentre randomised controlled BeBoP trial . ThroughThe BeBoP trial included participants (18 ≥ years) with diabetes mellitus and DFO in several sites in The Netherlands and Australia, from which, participant data from the Amsterdam University Medical Centres, locations Meibergdreef and De Boelelaan, were used for the present study.We included participants with suspected DFO, based on a positive probe-to-bone test, abnormalities on plain X-ray suggestive of osteomyelitis , erythro2). Besides these aerobic cultures, all specimens were also inoculated under anaerobic conditions, i.e., inoculation of bacteria on COS at 35–37 °C for 4 days. Specimens were inoculated in brain heart infusion broth (BHI) for 7 days to increase sensitivity. If these cultures showed bacterial growth, they were further inoculated on PVX and COS at 35–37 °C with CO2 under both aerobic and anaerobic conditions.All participants underwent an aseptically obtained percutaneous bone biopsy, adjacent to the ulcer through intact sterilised skin and a biopsy of the ulcer bed at inclusion. Specimens were examined using conventional culturing techniques for bacteria, according to standard operating procedures of the Amsterdam University Medical Centres laboratory. A Gram stain was performed and bacteria were inoculated on Columbia agar + 5% sheep blood (COS) and chocolate agar (PVX) at 35–37 °C under aerobic conditions with carbon dioxide . In our database, we scored whether a participant received antibiotics within the given timeframe of 2 months up to 7 days before biopsies, as well as the type of antibiotic agents, the number of different antibiotic agents, and the number of antibiotic courses. We excluded a course of nitrofurantoin and a single dose of oral fosfomycin, prescribed for uncomplicated urinary tract infection. These antibiotics do not reach therapeutic levels in the skin or bone. We also scored if a participant received prior antibiotics up to exactly 7 days before the acquisition of cultures or if the antibiotic-free period was longer. We created this variable to investigate if there is a difference between the risk of antibiotics administered shortly before biopsy or at a longer period of time prior.We collected participant data regarding antibiotic consumption from 2 months up to 7 days prior to the acquisition of ulcer bed and bone biopsies from: 1. The Electronic Health Record (EHR) of Amsterdam University Medical Centres; 2. The National Pharmacy Database . A negative culture was defined as the complete absence of growth, and a positive one included any bacterial growth, including the growth of low-virulence bacteria such as coagulase-negative staphylococci, S. aureus and S. lugdunensis, beta-haemolytic streptococci, Enterobacterales, and Pseudomonas spp. to one or more key antibiotic(s) for the treatment of DFO.The microbiologist determined if the combined results of ulcer bed and bone cultures of one participant were either susceptible  or resistant . We investigated the occurrence of resistance of common virulent (pathogenic) bacteria in DFO: S. aureus, S. lugdunensis, and beta-haemolytic streptococci;Clindamycin-resistance in Enterobacterales and Pseudomonas spp.;Ciprofloxacin-resistance in Enterobacterales;Trimethoprim/sulfamethoxazole-resistance in Multiple drug resistance (MDR), defined as acquired resistance to at least one agent in three or more antimicrobial classes .Resistance to key antibiotics was defined as:According to the new definition of S/I/R by EUCAST, isolates that are tested in the ‘I’ category are considered susceptible  .Corynebacterium spp., coagulase-negative staphylococci, and Enterococcus spp.We explored the occurrence of intrinsically resistant bacteria in cultures after the administration of prior antibiotics (pre-exposed vs. not pre-exposed participants): To investigate if prior antibiotics increased the risk of sterile cultures or increased the risk of culturing resistant bacteria in the cultures, we calculated relative risks with 95% confidence intervals. We calculated relative risks for the outcomes: 1. at least one sterile culture, 2. bone culture negative, 3. ulcer bed culture negative, 4. both cultures are negative, 5. resistant bacteria/a resistant bacterium in the cultures. We combined the obtained relative risks and rates of uncertainty with an estimation of clinical relevance. Additionally, we performed logistic regression analyses to investigate the influence of a shorter (7 days before acquisition of specimens) compared to a longer (>7 days but <2 months before acquisition of specimens) antibiotic-free interval. To investigate this, we created a categorical variable with three categories  and added this to the model. We also investigated the influence of the number of antibiotic courses prior to biopsies. Therefore, we created a categorical variable: 0 courses, 1 course, or 2 courses, and added this to the logistic regression model.


This Special Issue is addressed to practising engineers and researchers involved in developing reinforced concrete and masonry structures. The common areas are designing and researching. Our review of 24 published papers confirms our thesis about problems which can be solved with modern methods using advanced finite element models (FEMs). Although the articles cover many topics, they can be classified into four groups: research scope, design aspect, nature of research, and methods. As each of the papers mentioned in a particular group has its own significance and focuses on other areas of research, each group was divided into sections. The research scope group was divided into materials and structures; the design aspect group was divided according to design methods; the nature of research group was divided according to places that research was conducted, and the methods group was divided into numerical and parametric methods. Reinforced concrete and masonry structures were analysed separately. Common aspects of research and analysis were found for each type of structure. The employed new research, analytical, and design tools can be an inspiration and a milestone for further research activities.Masonry and reinforced concrete are the most common materials used to build structures and buildings. Development in the fields of material and construction solutions, modelling, and design methods has been dynamic in recent years. There is a tendency towards increasing the slenderness of masonry structures, improving strength parameters, and increasing sound and thermal insulation. New technological solutions do not only refer to masonry units and mortar, but also internal reinforcement, superficial reinforcement, and even prestressing reinforcement. A special type of high-strength concrete, the aim of reducing shrinkage, and the use of both dispersed and structural non-metallic reinforcement, have all been used in reinforced concrete structures. Considerable progress has been achieved in design methods, predicting structure durability and safety. This Special Issue on Masonry Structures and Reinforced Concrete Structures focuses on new material and structural solutions for masonry and reinforced concrete, and also includes laboratory tests, theoretical analyses, and numerical simulations. We planned to publish at least ten papers: five on reinforced concrete and five on masonry structures. Ultimately, 24 papers were published ,22,23,24This Special Issue includes 24 papers, of which 23 are original research projects, and 1 is a review; this Special Issue does not contain any technical reports. The published papers were prepared by 63 authors and co-authors from 10 countries: Saudi Arabia, Austria, China, Croatia, Egypt, France, Pakistan, Poland, Portugal, Romania, and Italy. Overall, 60% of the authors of papers on reinforced concrete structures represented technical universities. There are 1233 cited works in total. Although the research areas of the papers were specified, the aspects of reinforced concrete and masonry structures cover many topics. The scope of the published papers, relationships between mentioned topics, and the various aspects and applications of test results can be sorted into several groups, as illustrated in The order in which the papers were chosen to be presented was based on the subjective decision that papers on the properties of concrete components should be presented in first, followed by papers on tests on structural components and joints and finally the papers discussing the repair and strengthening of structures.The paper  by Li, K3 of synthetic fibres with different geometry and form was presented. The flexural tensile strength of fibre-reinforced concrete was analysed during a three-point bending test. The results obtained from three-point bending tests significantly differed from empirical equations for calculating the flexural tensile strength of fibre-reinforced concrete with dispersed steel fibres present in the literature. The authors suggested a test-based equation for determining the strength of concrete with polymer fibres with a nominal fibre content ≤ 1.0% and slenderness of up to 200. This equation is also applicable to the satisfactory determination of fibre-reinforced concrete for studies performed by other researchers.Tests on the strength of fibre-reinforced concrete were described by Blazy, Drobiec, and Wolka in the paper . The posAspects of concrete ageing in mass structures were considered as both material and structural problems. This topic was presented in two papers ,18. The l1 = 0.582% and ρl2 = 2.332%). The authors also analysed the cracking and failure mechanism of beams, and load-carrying capacity and displacements with a new type of truss reinforcement. The obtained results were compared with beams with conventional reinforcement. Truss reinforcement was found to increase the load-carrying capacity of beams depending on the ratio of longitudinal reinforcement. Beams with a reinforcement ratio of (0.582%) showed an increase in load-carrying capacity of 95%, and beams with a reinforcement ratio of 2.332% showed an increase in load-carrying capacity of 12%. Failure of beams with truss reinforcement was observed when a greater number of smaller cracks were arranged uniformly over the area of pure bending, which resulted in a smaller range and narrower width. The comparative analysis showed high effectiveness of truss reinforcement, which confirmed potential applications for reinforcing structural elements. And the paper [Tests on reinforced concrete beams are described in the papers ,20, wherThe paper  by Jancy1 ≤ 0.5, no concentration of internal forces in corners was observed. The simplified method specified in the Model Code 2010 standard was found to give a considerably lower effective length of control perimeter for high values of thickening/widening of flat slabs when compared to the more accurate method.Theoretical tests on slab and column systems are described by Grabski and Ambroziak in the paper . The effKorentz, in the paper , analyseGołdyn and Urban, in the paper , focusedThe papers ,17,19 ca3. Stiffness and a number of temporary supports built into the structure during rectification were analysed. Stiffness of these supports was determined during in situ tests. Then, the model of the rectified structure was defined and its parameters expressed using three matrices: stiffness, displacement modes of the elevated tank, and displacement modes of supports. The conducted electricity balance demonstrated that the amount of energy required for the vertical displacement of the construction decreased with increasing stiffness of the supports and the number of supports. This meant that a greater number of supports under rectified structures and stiffer supports was desirable for the rectification process. Results from the conducted analyses were verified during the in situ tests.A unique method of repairing buildings was presented by Gromysz in the paper . Based oSimilarly to reinforced concrete structures, the papers on masonry structures are organised as follows: firstly, papers on the properties of masonry components are presented; followed by those regarding tests on masonry, walls, or structures are presented; and finally, the papers on the repair and strengthening of masonry structures and their reliability are listed.The tests on mortar types used in wall construction are presented in the papers ,7,23.Łątka, in the paper , describKubica and Galman  presenteTests on masonry units are presented in the paper . Abbass,The aspects of strengthening were described in the papers ,13,24. TThe tests on elements and structures are presented in three papers ,9,21. InKozłowski, Galman, and Jasiński, in the paper , describγM for masonry. They demonstrated that safety factors were much lower than those specified in the standard PN-EN 1996-1-1.Zięba, Skrzypczak, and Buda-Ożóg, in the paper , dealt wEach of the published papers can be categorized into the “Research scope” group—as shown in However, one should be aware that technical progress in research alone will not solve the design problems of reinforced concrete or masonry structures. This is a more complex problem involving many factors, including legal ones. These facts create many challenges for engineers and researchers in the scope of their testing and analysing with new methods.This review of 24 published papers shows that the range of tests on reinforced concrete and masonry structures is multi-dimensional. There are still many topics which have not yet been tested or analysed. Both tests and analyses of reinforced concrete and masonry structures were conducted using optical techniques for measuring (DIC) and advanced numerical models (FEM). Moreover, there are many common and complementary aspects concerning material (component elements) tests and strengthening. It is obvious that modern methods of testing and analysing can be an incentive to solve known problems with new methods. This is particularly important for the application of pioneering materials whose manufacturing processes take into account environmental protection and ecology.Design methods, which are always the subject of tests and research inquiries, are continuously evolving. The papers discussed herein put an emphasis on the fact that new analytical tools can combine subjectively different aspects.


The control concrete was made from quartz sand and tap water, and the sea concrete was made from sea water and sea sand for the purpose of applying local building materials to construction sites in the coastal area. The properties of a self-compacting concrete mixture, as well as concrete and textile-reinforced concrete based on it, were determined. It was found that at the age of 28 days, the compressive strength of the sea concrete was 72 MPa, and the flexural strength was 9.2 MPa. The compressive strength of the control concrete was 69.4 MPa at the age of 28 days, and the flexural strength was 11.1 MPa. The drying shrinkage of the sea concrete at 28 days exceeded the drying shrinkage of the control concrete by 18%. The uniaxial tensile test showed the same behavior of the control and marine textile-reinforced concrete; after the formation of five cracks, only the carbon textile reinforcement came into operation. Accordingly, the use of sea water and sea sand in combination with a cement with reduced CO The minimum amount of water to ensure cement hydration is about 25% of the cement weight [Today, concrete remains the most popular building material in the world . In addiIt is worth noting that only about 2.5% of the world’s ocean water is fresh water, and about 40% of the world’s population lives within 100 km of the sea coast . Moreove2 in terms of climate impact. This is a measurement scale developed and implemented by the United Nations’ Intergovernmental Panel on Climate Change [2 equivalents are also referred to as global warming potential (GWP) and abbreviated to CO2-eq in mass units (kg). For a product , these values can be found in its respective environmental product declaration (EPD), which is defined by ISO 14025 [Today, it is widely recognized that concrete is one of the greatest contributors to global warming, and different strategies to produce environmentally efficient concretes already exist and are implemented in practice. Its environmental sustainability can be assessed on the basis of the European standard EN 15978 [e Change  to repreSO 14025 . The comSO 14025 .Lately, the material technology has offered various possibilities for the replacement of cement with other binders, such as calcium sulphate aluminates (CSA) and geopolymers, which can lead to a relative reduction of approximately 30% or 80%, respectively . Replaci2,e/ton compared to the extraction from pits and quarries with 0.276 and 0.737 kg CO2,e/ton, respectively [Sand has for a long time been considered an easily accessible and seemingly abundant raw material, which can be extracted from mines and deposits, including deserts. Vast quantities, in the range of 50 billion tons annually, are consumed globally in the production of various industrial products, such as glass, filters, microchips/electronics, shale oil from fracking, but also in construction as a filler or a concrete aggregate . In receectively . 3 of water on earth, only 2.5% is drinkable fresh water, 0.3% of which is directly accessible via rivers, streams or lakes on the continental surface [Regarding water, as another significant constituent of concrete, out of the 1.4 billion km surface . At the  surface  indicate surface . As withIn experiments ,24,25,26It should be noted that for construction in the coastal zone, it is important to ensure sufficient sulphate resistance of concrete structures, according to . A separ2 emissions through the use of sea textile-reinforced concrete.Textile-reinforced concrete (TRC) contains a rectangular arrangement of fiber bundles made of non-corroding materials, such as carbon, glass or basalt. Textile reinforcement is produced from continuous fibers (filaments) in the form of rovings, which can contain more than 40,000 individual filaments. Textile reinforcement has close-meshed openings, which increase the ductility of concrete. Stresses and cracks are distributed over a larger area, thereby reducing cracking in the structure elements. The smaller diameter of the cross-section of textile reinforcement (1–2 mm) allows to reduce the thickness of structures and consequently reduce their total weight ,40. A fe3) for the control concrete mixture and Dyckerhoff CEM III/B 42.5 N-LH/SR (bulk density = 1180 kg/m3) for the sea concrete mixture. The use of CEM III/B 42.5 cement results in a 47% reduction in CO2 emissions (CEM I 42.5 N/CEM III/B 42.5 N-LH/SR = 661/311 kg CO2-Eq/t). Normal and sea sand were used as fine aggregates; the particle size distribution was determined according to Ref. [3; using https://reefapp.net/de/salinity-calculator (accessed on 23 April 2023), this corresponds to a salinity of 3.5%.Two types of cement were used to prepare self-compacting concretes: Dyckerhoff CEM I 42.5 N , 2 plates with a size of 1300 mm × 420 mm × 10 mm were produced based on the compositions of self-compacting concrete from First, a 6 mm layer of concrete was placed; then, the carbon mesh was laid and slightly troweled; then, a second layer of 4 mm concrete mixture was placed and then troweled . The man-compressive strength at age of 3, 7 and 28 days according to the methodology in , load ap-flexural strength at age of 3, 7 and 28 days according to the methodology in  . This may be due to the high air content of the fresh concrete mixture and the consequent formation of more open pores on the surface of the hardened concrete. Additionally, refs. ,59,60 co3A, which contributes significantly to early strength development. At the age of 7 days, the difference in compressive strength decreased to 2%—65 MPa for control concrete and 63.7 MPa for sea concrete. At the design age, the strength of sea concrete exceeded the strength of control concrete by 3.7% and amounted to 72 MPa. Six samples for each batch were used for compressive strength tests. The compressive strength of the control self-compacting concrete at the age of 3 days was expectedly higher than the compressive strength of sea self-compacting concrete; the difference was 29.2%. This is due to the fact that the sea concrete was made on a CEM III/B 42.5 N-LH/SR base with a low clinker content, and consequently, a lower content of tricalcium aluminate CThe authors in ,63,64 coThe data obtained on the flexural strength of the studied self-compacting concretes are shown in the diagram in The modulus of elasticity of concrete is an important parameter, which is used in the calculation of building structures and characterizes the ability of concrete to retain its elastic properties under external load. c at the age of 28 days was 39.1 GPa, and for the control concrete, it was 38.9 GPa. This is due to the lower compressive strength of the control concrete composition. Other studies [For sea concrete, the modulus of elasticity E studies ,68 also Shrinkage is one of the most important parameters for the durability of concrete. In this study, the drying shrinkage was determined using the linear method . The tensile tests were carried out according to the load application scheme shown in The diagram shows that the tensile strength of sea concrete was 3.6 MPa, and the tensile strength of control concrete was 3.3 MPa. This area on the diagram has a linear character. With the development of the first crack, textile reinforcement is included in the work, and the stage of multiple cracking begins—a sawtooth section in the diagram. It was experimentally established that the number of cracks on both types of concrete was five pieces. The average distance between cracks on the samples of sea TRC was 9.6 cm, and on the samples of control TRC, the distance was 10 cm. With a further increase in the load, the bond between the reinforcing mesh and concrete is broken, and only textile reinforcement continues to work.The absorption energy of sea and control TRC was also calculated for specimen displacement at 10 mm. For this purpose, the average area (three samples) under the stress–strain curve for each TRC type was determined. For the sea TRC composition, the fracture energy was 874.56 kJ, and for the control TRC composition, it was 835.12 kJ. Thus, 4.7% more energy was required to achieve a displacement length of 10 mm for the sea TRC compared to the control TRC.The obtained data on the performance of textile-reinforced concrete are consistent with the research ,42,69. S2 emissions per 1 m3 of concrete in the extraction and production stages (A1–A3), according to [Since the purpose of this study was to determine not only the physical and mechanical properties of concretes but also their impact on the environment, this section determined the amounts of COrding to , which arding to ,73,74,752 emissions by 91.5% compared to the control SCHSC. In turn, the use of slag cement has the most impact on decreasing CO2 emissions. Additionally, the use of sea sand up to 50 km from the coast would generate much less emissions than the transportation of sand from a quarry.The data show that the use of Type III low clinker cement, sea sand and sea water for SCHSC production can reduce CO2 emissions are recorded at the component level compared to steel in the following research works [A direct comparison between steel reinforcement and textile reinforcement with carbon rovings via their global warming potential does not seem appropriate due to an energy-intensive process. However, it should be considered that carbon reinforcement is much lighter in weight, in addition to the fact that the bearing capacity can be up to six times higher . There ich works ,78,79.-the flowability of the sea concrete was more viscous than the control concrete because of the amount of sea shells, which are contained in the sea sand, and because of the salt in the sea water, which accelerated the hydration. Therefore, a higher content of superplasticizer for the concrete mixture with sea water and sea sand can be expected. Additionally, the air content test confirmed that, although self-compacting concrete was produced, which usually has a higher air content, in this case, it was below the limit of 5–6% and had no negative influence on the pore structure;-the use of sea sand and sea water for the production of SCHSC based on slag cement makes it possible to produce concretes with comparable mechanical properties while significantly improving the life cycle assessment. At the age of 3 days, the compressive strength of the sea concrete composition was 37.3 MPa, which was around 10.9 MPa lower than the compressive strength of the control concrete composition at 48.2 MPa, due to the slower hydration rate of the slag cement. However, this cannot be considered a negative effect because, in new construction, early strength is not paramount. It was experimentally established that at the age of 28 days, sea concrete had a compressive strength of 72 MPa, which corresponds to the concrete grade of C45/55. The strength of the control concrete was 69.4 MPa, which corresponds to the concrete grade of C40/45. The flexural strength at the age of 3, 7, 28 days of sea concrete was slightly lower  than the flexural strength  of control concrete due to the weak surface contact between the shells and the cement matrix;-after 28 days, the modulus of elasticity for the sea concrete showed a higher value of 39.1 GPa in comparison to the control concrete value of 38.9 GPa, which showed that sea water had no effect on it;-the studies on durability in the form of drying shrinkage show that at the early ages (3 days), the sea concrete shrinkage deformations exceed 122% with 0.6 mm/m in comparison to the control concrete. This is explained by the fact that, with the use of sea water, the pore structure is refined, and the capillary pores (>100 nm) are reduced . Due to -the combination effect of sea concrete as an SCHSC with textile reinforcement on the tensile strength (3.6 MPa) shows equal results to the control concrete (3.3 MPa) with a slightly increasing trend. The obtained data on the performance of textile-reinforced concrete based on sea and control concrete matrices show the same result.-2 emissions can be significantly reduced. The ability to use sea sand as an aggregate also eliminates the need to deliver this material to the construction site, which further reduces CO2 emissions. 2 emissions for 1 m3 of concrete would be more than 90% in total. In addition, the use of textile-reinforced concrete allows us to reduce the cross-sectional thickness of structural elements, which makes it possible to reduce the material consumption and bypass corrosion of steel reinforcement due to sea sand/water.the raw materials used for the production of concrete in our experiment can be recommended for the construction of facilities near the sea coast, which makes it possible to obtain SCHSC. It is important to note that by using slag cement in the production of textile-reinforced structures, COThis paper provides an overview of the use of sea water and sea sand in combination with textile reinforcement with the following conclusions:Further research should be directed to the study of the microstructure of sea concretes. Of particular interest is the study of the effect of different salinities of sea sand and sea water on the properties of sea concrete. The possibility of reducing shrinkage deformations of sea concrete by using dispersion reinforcement must also be considered. Special attention should be paid to durability indicators, such as frost resistance, sulphate resistance and water permeability. This will expand the existing data and, in the future, will allow the use of concrete based on slag cement, sea water and sea sand not only for residential construction in the coastal zone but also for the construction of coastal protection and offshore structures.


Pinellia ternata. In this study, the effects of chitosan on the growth, photosynthesis, resistance, yield, and quality of the continuous cropping of P. ternata were investigated by two field spraying methods. The results indicate that continuous cropping significantly (p < 0.05) raised the inverted seedling rate of P. ternata and inhibited its growth, yield, and quality. Spraying of 0.5~1.0% chitosan effectively increased the leaf area and plant height of continuous P. ternata, and reduced its inverted seedling rate. Meanwhile, 0.5~1.0% chitosan spraying could notably increase its photosynthetic rate (Pn), intercellular carbon dioxide concentration (Ci), stomatal conductance (Gs), and transpiration rate (Tr), and decrease its soluble sugar, proline (Pro), and malonaldehyde (MDA) contents, as well as promoting its superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities. Additionally, 0.5~1.0% chitosan spraying could also effectively enhance its yield and quality. This finding highlights that chitosan can be proposed as an alternative and practicable mitigator for alleviating the continuous cropping obstacle of P. ternata.The continuous cropping obstacle has become the key factor that seriously restricts the growth, yield, and quality of  Pinellia ternata (Thunb.) Breit., a medical and ornamental perennial herb of Pinellia in the Araceae family, has been widely planted in northwest and southwest China [P. ternata industry has flourished rapidly in Guizhou and Gansu Provinces in China, with cropping areas of over 4000 and 1333 hm2, respectively, and made an energetic contribution to alleviating poverty and revitalizing rural areas [P. ternata, the cultivation measures to promote its growth, yield, and medicinal quality have attracted increasing attention.st China . It is rst China ,3,4. As st China ,6,7,8. RP. ternata, its growth and development are inhibited, its quality deteriorates, its susceptibility to diseases and pests is intensified, and its yield declines [P. ternata by increasing fertilizer use and abusing pesticides, which also leads to problems such as agricultural product quality safety and environmental pollution. Thus, the continuous cropping obstacle has become the key factor that seriously restricts the growth, quality, yield and development of P. ternata, and the establishment of its prevention and control strategy has become a hot issue in research. Currently, soil disinfection, intercropping, and rotation measures are often used to alleviate the continuous cropping obstacle of P. ternata [P. ternata. Hang et al. [P. ternata, and intercropping pepper could enhance the succinic acid and guanosine contents of P. ternata. However, the application and promotion of these measures are limited by factors including operability, low efficiency, and environmental pollution, etc. Considering the serious harmfulness of the continuous cropping obstacle, more alternative and practicable control measures need to be developed for enhancing the healthy development of the P. ternata industry.Unfortunately, after continuous cropping of declines ,10,11,12declines ,14. Addi ternata ,14,15. F ternata  reportedN-deacetylation product of chitin, has been widely applied in food, agriculture, medicine, and cosmetic fields due to its advantages including nontoxicity, bioactivity, biodegradability, and renewability [Rosa roxburghii resistance, growth, yield, and quality. Pan et al. [Trifolium repens under salt stress. Li et al. [Sctellaria baicalensis seedlings. Recently, Liu et al. [Platycodon grandifloras. So far, there has been no reports in the literature of whether chitosan promotes the growth of P. ternata. Accordingly, whether chitosan can enhance the growth, resistance, yield, and quality of continuous P. ternata, and alleviate its continuous cropping obstacle, is worth further study.Chitosan, a natural, nontoxic to humans or other organisms, wability ,17,18,19wability ,22,23,24wability ,26,27,28wability  reportedn et al.  indicatei et al.  reportedu et al.  indicateP. ternata was evaluated. Simultaneously, the stress resistance capacity of plants strongly affects their growth; soluble sugar, proline (Pro), malonaldehyde (MDA), superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) are all closely connected to stress resistance. Thus, the influences of chitosan on the abovementioned resistance parameters of continuous P. ternata was assessed. Finally, the influences of chitosan spraying on its yield and quality were also investigated. The aim of this study is to provide a new and alternative strategy for alleviating the continuous cropping obstacle of P. ternata.In this work, the influence of chitosan on the growth  and photosynthesis , intercellular carbon dioxide concentration (Ci), stomatal conductance (Gs), and transpiration rate (Tr)) of the continuous P. ternata plants is shown in P. ternata for 2 years, at the vigorous growth and inverted seedling periods, was significantly (p < 0.05) inferior to that of non-continuous P. ternata; and the plant height at the full seedling, vigorous growth, and inverted seedling periods was also significantly (p < 0.05) inferior to that of non-continuous P. ternata; the stem diameter at the three growth periods was also lower than that of non-continuous P. ternata. Compared with plants not sprayed with chitosan, 0.5~1.0% chitosan spraying significantly (p < 0.05) enhanced the leaf area of continuous P. ternata plants at the vigorous growth and inverted seedling periods, and the plant height of continuous P. ternata plants at the full seedling, vigorous growth, and inverted seedling periods. Additionally, compared with non-continuous P. ternata, the leaf area, plant height, and stem diameter of continuous P. ternata plants treated by 0.1~1.0% chitosan spraying were still inferior. The present results indicate that the continuous cropping significantly (p < 0.05) decreased the leaf area, plant height, and stem diameter of P. ternata plants, and that 0.5~1.0% chitosan spraying had a promoting effect on the growth of continuous P. ternata plants.The influence of chitosan spraying on the leaf area, plant height, and stem diameter of P. ternata, which is a dormancy to resist the adverse environment. During the growth of P. ternata, when the environmental conditions such as temperature, humidity, light, allelochemicals, etc. change greatly, its aboveground stems and leaves will gradually wither, and it makes underground stems (inverted seedling) to survive the adverse environment. The influence of chitosan on the inverted seedling rate of P. ternata plants is displayed in p < 0.05) increased the inverted seedling rate of P. ternata plants, 0.5~1.0% chitosan spraying effectively (p < 0.05) decreased the inverted seedling rate of the continuous P. ternata plants. However, the inverted seedling rate in plants treated by 0.1~1.0% chitosan significantly (p < 0.05) exceeded that of non-continuous P. ternata plants. These findings emphasize that 0.5~1.0% chitosan spraying could effectively decrease the inverted seedling rate of P. ternata, and alleviate its cropping obstacle in part, but not completely, which might be related to chitosan application enhancing the stress resistance of P. ternata.The inverted seedling is a physiological phenomenon that often occurs during the growth of P. ternata leaves treated by 0.1~1.0% chitosan spraying was lower than that of non-continuous P. ternata leaves, and the continuous cropping or chitosan treatment had no significant influence on the P. ternata chlorophyll content. The influences of chitosan spraying on the Pn, Ci, Gs, and Tr contents of P. ternata are shown in P. ternata were significantly (p < 0.05) lower than those of the non-continuous P. ternata. Compared with plants not sprayed with chitosan, 0.5~1.0% chitosan spraying significantly (p < 0.05) enhanced the Pn and Ci contents of the continuous P. ternata, and 0.1~1.0% chitosan spraying significantly (p < 0.05) enhanced the Gs and Tr contents. Meanwhile, the Pn, Ci, Gs, and Tr contents of the continuous P. ternata treated by 0.1~1.0% chitosan spraying were significantly (p < 0.05) smaller than those of the non-continuous P. ternata. The results presented here indicate that the foliar application of 0.5~1.0% chitosan spraying effectively increased the Pn, Ci, Gs, and Tr contents of the continuous P. ternata, thereby promoting favorable growth.As shown in P. ternata plants is displayed in p < 0.05) raised the soluble sugar, Pro, and MDA contents of P. ternata compared with non-continuous cropping, and decreased the SOD, POD, and CAT activities. Compared with non-sprayed chitosan, 0.1~1.0% chitosan spraying significantly (p < 0.05) decreased the soluble sugar, Pro, and MDA contents of the continuous P. ternata, and 0.5~1.0% chitosan spraying significantly (p < 0.05) enhanced its SOD, POD, and CAT activities. Meanwhile, the soluble sugar, Pro, and MDA contents of the continuous P. ternata treated by 0.1~1.0% chitosan spraying were significantly (p < 0.05) higher than those of non-continuous P. ternata, and its SOD, POD, and CAT activities were significantly (p < 0.05) inferior to those of non-continuous P. ternata. These results show that continuous cropping significantly (p < 0.05) decreased the stress resistance of P. ternata plants, and 0.5~1.0% chitosan spraying could effectively decrease the soluble sugar, Pro, and MDA contents of continuous P. ternata and promote its SOD, POD, and CAT activities, thereby enhancing the stress resistance of continuous P. ternata.The influence of chitosan spraying on the soluble sugar, Pro, MDA, SOD activity, POD activity, and CAT activity of p < 0.05) decreased the seed weight, medicinal material weight, and total yield of P. ternata, compared with the non-continuous cropping plants. Spraying with 0.1~1.0% chitosan led to an increase in the yield for the continuous P. ternata, with the mean seed weight, medicinal material weight, and total yield of 27.56–46.86, 85.69–158.52, and 113.25–205.38 kg per 667 m2, respectively, significantly (p < 0.05) higher ((1.17–1.99)-fold, (1.09–2.01)-fold, (1.10–2.00)-fold, respectively) than for the non-sprayed plants. Meanwhile, the seed weight, medicinal material weight, and total yield of the continuous P. ternata treated by 0.1~1.0% chitosan spray were still significantly (p < 0.05) lower than those of the non-continuous P. ternata. The results indicate that 0.1~1.0% chitosan spraying could effectively alleviate the cropping obstacle of P. ternata and enhance its bulb growth and yield formation.As shown in P. ternata is shown in p < 0.05) reduced the water, ash, extractum, and succinic acid contents of P. ternata compared with non-continuous cropping plants. Compared with plants not sprayed with chitosan, 0.5~1.0% chitosan spraying significantly (p < 0.05) increased the water, ash, extractum, and succinic acid contents of continuous P. ternata. However, the water, ash, extractum, and succinic acid contents of continuous P. ternata treated by 0.5~1.0% chitosan spraying were still significantly (p < 0.05) lower than those of non-continuous P. ternata. These findings indicate that 0.5~1.0% chitosan spraying effectively improved the medicinal quality of continuous P. ternata bulbs.The influence of chitosan spraying on the water, ash, extractum, and succinic acid of P. ternata [P. ternata, and continuous cropping for 2 years exhibited a stronger inhibitory effect compared with continuous cropping for 1, 3, 4, and 5 years. In this study, P. ternata in the continuous cropping group for 2 years was used as the treatment object, and P. ternata in the non-continuous cropping group was used as the intergroup control. The results indicate that continuous cropping for 2 years significantly (p < 0.05) increased the inverted seedling rate of P. ternata and inhibited its growth, yield, and quality, and caused it physiological damage and put it in a state of stress, which was consistent with previous results. Chitosan can promote cell division and elongation, via activating the gene expression and signal transduction of cytokinin and auxin, which enhance the nutrient intake and growth of plants [Trifolium repens under salt stress. Liu et al. [Platycodon grandifloras. In this work, 0.5~1.0% chitosan spraying significantly (p < 0.05) increased the leaf area and plant height of continuous P. ternata, and decreased its inverted seedling rate. These results emphasize that 0.5~1.0% chitosan spraying could effectively alleviate the cropping obstacle of P. ternata.The continuous cropping obstacle seriously restricts the growth, quality, yield and development of  ternata ,12,13,14 ternata  found thf plants ,23,24,25f plants  found thu et al.  reportedPlatycodon grandifloras photosynthesis. Our previous research also showed that chitosan spraying could promote the photosynthesis of Actinidia chinensis and Rosa roxburghii [p < 0.05) increase the chlorophyll content of the continuous P. ternata, 0.5~1.0% chitosan spraying could effectively enhance its Pn, Ci, Gs, and Tr contents, which further highlights that chitosan spraying could effectively alleviate the cropping obstacle of P. ternata. The osmotic potential of plant tissue under stress can be regulated by soluble sugar, to maintain plant growth, and Pro can effectively protect the structural integrity of cell films, while MDA is an indicator of membrane damage and stress resistance degrees [2O2 decomposition in lignin biosynthesis, as such they are connected to stress resistance [Sctellaria baicalensis seedlings, and Dzung et al. [Coffea spp., as well as the disease resistance of Actinidia chinensis and Rosa roxburghii [p < 0.05) increased the soluble sugar, Pro, and MDA contents of P. ternata, and decreased its SOD, POD, and CAT activities, as well as put it in a state of stress. Effectively, 0.5~1.0% chitosan spraying could significantly (p < 0.05) decrease the soluble sugar, Pro, and MDA contents of the continuous P. ternata, and promote its SOD, POD, and CAT activities, thereby enhancing the stress resistance of the continuous P. ternata. These results also demonstrate that 0.5~1.0% chitosan has a notably alleviatory effect on the cropping obstacle of P. ternata.Photosynthesis is the physiological basis of plant life action, while chitosan is an enhancer of photosynthesis . Liu et xburghii ,35. In t degrees . SOD andsistance . Li et asistance  found thg et al.  indicatexburghii ,37,38. IP. ternata. Pan et al. [Trifolium repens under salt stress. Liu et al. [Platycodon grandifloras, and Li et al. [Rosa roxburghii. In this study, continuous P. ternata sprayed with 0.1~1.0% chitosan exhibited an increased yield, with the seed weight, medicinal material weight, and total yield. Spraying with 0.5~1.0% chitosan effectively enhanced the water, ash, extractum, and succinic acid of continuous P. ternata bulbs. These results emphasize that chitosan is an effective mitigator for alleviating the continuous cropping obstacle of P. ternata. However, the growth, photosynthesis, resistance, yield, and quality of the continuous P. ternata treated by 0.5~1.0% chitosan spraying were still significantly (p < 0.05) inferior to those of non-continuous P. ternata. This finding highlights that 0.5~1.0% chitosan spraying could effectively alleviate the cropping obstacle of P. ternata, but cannot completely remove it.Good growth is the basis for the high yield and superior quality of n et al.  indicateu et al.  reportedi et al. ,29 also P. ternata is as follows: chitosan might be used as a plant growth promoter or biological fertilizer to enhance the growth and photosynthesis of continuous P. ternata, and might also be used as an inducer to improve its stress resistance and reduce its inverted seedling rate, as well as might also be used as a biological fungicide to prevent disease occurrence. These possible positive effects together, enhanced the growth, yield, and quality of continuous P. ternata and alleviated its continuous cropping obstacle. In the future, the molecular biological mechanism of the chitosan-mediated alleviation of the continuous cropping obstacle of P. ternata needs to be studied. Meanwhile, chitosan is a natural biopolymer, with nontoxic, renewable, and biodegradable advantages [P. ternata was very long. Accordingly, the safety risk caused by chitosan was almost nonexistent. This work emphasizes that chitosan can be proposed as a practicable promotor or mitigator for promoting the growth, photosynthesis, resistance, yield, and quality of continuous P. ternata, and alleviating its continuous cropping obstacle.Overall, the possible mechanism of how chitosan mediated the alleviation of the continuous cropping obstacle of vantages ,20,37, aP. ternata (diameter of 0.6~1.2 cm) were provided by the Institute of Modern Chinese Herbal Medicines, Guizhou Academy of Agricultural Sciences . Other chemicals were analytical or chromatographic grade.Water-soluble chitosan (deacetylation ≥ 90.00%) was produced by Mingrui Bioengineering Co., Ltd. . Seed bulbs of ball P. ternata herb garden was divided into two parts: One part had never been cultivated with P. ternata , and the other part had been cultivated with P. ternata in the past two years. In the third year, the chitosan spraying experiment was carried out, and the experimental groups included the no continuous cropping group and the continuous cropping for 2 years group. P. ternata was cultivated by ridging, and each treated plot area was 3.6 m2 . Seed bulbs of ball P. ternata were broadcast-sown on 15 March 2022, then covered with 5 cm of soil, and the application dosage of the seed bulbs was 140 kg per 667 m2. The basic information of the P. ternata herb garden is shown in The P. ternata treated by clear water , (2) continuous P. ternata treated by 0.1% chitosan dilution liquid (Ch 0.1), (3) continuous P. ternata treated by 0.5% chitosan dilution liquid (Ch 0.5), (4) continuous P. ternata treated by 1.0% chitosan dilution liquid (Ch 1.0), and (5) non-continuous P. ternata treated by clear water . Each treatment had three replicates, with a total of 15 plots. The P. ternata plants were sprayed by chitosan dilution liquid using an electrostatic atomizer  on April 15 and April 30. The application dosage of the chitosan dilution liquid was 60 L per 667 m2.The foliar spray method and completely randomized method were used for spraying chitosan and delineating experimental plots, respectively. Five treatments were designed: (1) continuous P. ternata plants were monitored at full seedling period (April 30), vigorous growth period (May 15), and inverted seedling period (May 30). The stem diameter was measured by a vernier caliper, and plant height, and leaf length and width, were measured by a ruler. The leaf area coefficient method was used to calculate the main leaf area of P. ternata:The leaf area, height, and stem diameter of the P. ternata plants, on the east, west, south, north, and in the middle of each plot, were randomly selected at the vigorous growth period (May 15) for investigation of their photosynthesis parameters. The chlorophyll content of the fully-expanded leaves of P. ternata was monitored by a SPAD-502Plus chlorophyll meter . Meanwhile, their Pn, Ci, Gs, and Tr contents were monitored by a portable LI-6400XT photosynthesis measurement system , with the photosynthetically active radiation of 1000 μmol m−2 s−1 at 8:00–10:00 a.m. [Fifteen :00 a.m. ,28.P. ternata, such as soluble sugar, Pro, MDA, SOD activity, POD activity, and CAT activity, according to the methods of Wang et al. [The fresh plants with the same orientation were collected and taken back to the laboratory to determine the resistance indexes of g et al.  and Zhang et al. ,40. AnthP. ternata in each plot were collected and washed on September 10. Among them, those with a diameter greater than 1.2 cm were selected as medicinal materials, and those with a diameter less than 1.2 cm were reserved as seeds. Their weights were determined by the gravimetric method. The water, ash, extractum, and succinic acid of the P. ternata bulbs were determined as described by the general principles of four parts of Chinese Pharmacopoeia, 2020 [The underground bulbs of ia, 2020 .The data represent mean values ± standard deviation (SD) of three replicates. The Duncan’s test, with one-way analysis of variance (ANOVA), on the SPSS 18.0 software , was used for analyzing significant differences. The Origin 10.0 software  was used to create the figures.P. ternata and reduce its inverted seedling rate. Meanwhile, 0.5~1.0% chitosan spraying notably enhanced the photosynthesis and resistance of continuous P. ternata, and effectively improved its yield and quality. This study emphasizes that chitosan can be proposed as an enhancer or mitigator for promoting the growth, yield, and quality of continuous P. ternata, and alleviating its continuous cropping obstacle. However, 0.5~1.0% chitosan spraying could not completely alleviate the cropping obstacle of P. ternata. Thus, the molecular biological mechanism of chitosan for alleviating the continuous cropping obstacle of P. ternata, and the screening of its synergistic surfactant or adjuvant, need to be studied in the future.In conclusion, 0.5~1.0% chitosan spraying could effectively improve the leaf area and plant height of continuous


Clinical and preclinical studies indicate that adaptations in corticostriatal neurotransmission significantly contribute to heroin relapse vulnerability. In animal models, heroin self-administration and extinction produce cellular adaptations in both neurons and astrocytes within the nucleus accumbens (NA) core that are required for cue-induced heroin seeking. Specifically, decreased glutamate clearance and reduced association of perisynaptic astrocytic processes with NAcore synapses allow glutamate release from prelimbic (PrL) cortical terminals to engage synaptic and structural plasticity in NAcore medium spiny neurons. Normalizing astrocyte glutamate homeostasis with drugs like the antioxidant N-acetylcysteine (NAC) prevents cue-induced heroin seeking. Surprisingly, little is known about heroin-induced alterations in astrocytes or pyramidal neurons projecting to the NAcore in the PrL cortex (PrL-NAcore). Here, we observe functional adaptations in the PrL cortical astrocyte following heroin self-administration (SA) and extinction as measured by the electrophysiologically evoked plasmalemmal glutamate transporter 1 (GLT-1)-dependent current. We likewise observed the increased complexity of the glial fibrillary acidic protein (GFAP) cytoskeletal arbor and increased association of the astrocytic plasma membrane with synaptic markers following heroin SA and extinction training in the PrL cortex. Repeated treatment with NAC during extinction reversed both the enhanced astrocytic complexity and synaptic association. In PrL-NAcore neurons, heroin SA and extinction decreased the apical tuft dendritic spine density and enlarged dendritic spine head diameter in male Sprague–Dawley rats. Repeated NAC treatment during extinction prevented decreases in spine density but not dendritic spine head expansion. Moreover, heroin SA and extinction increased the co-registry of the GluA1 subunit of AMPA receptors in both the dendrite shaft and spine heads of PrL-NAcore neurons. Interestingly, the accumulation of GluA1 immunoreactivity in spine heads was further potentiated by NAC treatment during extinction. Finally, we show that the NAC treatment and elimination of thrombospondin 2 (TSP-2) block cue-induced heroin relapse. Taken together, our data reveal circuit-level adaptations in cortical dendritic spine morphology potentially linked to heroin-induced alterations in astrocyte complexity and association at the synapses. Additionally, these data demonstrate that NAC reverses PrL cortical heroin SA-and-extinction-induced adaptations in both astrocytes and corticostriatal neurons. Relapse remains a major clinical obstacle in treating opioid use disorders and is typically brought on by a craving evoked by exposure to opioid-associated contexts or cues . In humaThe nucleus accumbens core (NAcore) is a ventral striatal region required for motivationally relevant external stimuli to evoke motor programs linked to seeking reinforcers or rewards . It has One drug that is particularly effective at targeting addictive drug-induced adaptations in astrocytes is the antioxidant, N-acetylcysteine (NAC). NAC is an L-cysteine pro-drug that has shown some promise clinically  but has While there is a substantial body of work regarding the consequences of heroin SA and extinction on neuronal and astrocyte plasticity in the NAcore, little is known about the impact of heroin on neurons or astrocytes in the PrL cortex. This gap in our knowledge is particularly significant given that cue-induced glutamate release in the NAcore during heroin seeking requires THE activation of the PrL cortex . While lHere, we first employ electrophysiological recordings to interrogate functional adaptations in PrL astrocytes following heroin SA and extinction. Next, we employ immunohistochemical preparations in conjunction with confocal microscopy and 3D rendering to investigate the effects of heroin SA and extinction upon the GFAP cytoskeletal complexity of PrL astrocytes in addition to the PAP association with synaptic markers of NAcore projecting PrL neurons. We also investigate the therapeutic efficacy of NAC in reversing heroin SA-and-extinction-induced alterations in neuronal and astrocyte morphology. Finally, we investigate the effects of the inhibition of PrL thrombospondin 2 (TSP-2) in the context of NAC treatment following heroin SA, extinction, and cued-relapse to heroin seeking as a means to determine if NAC exerts its anti-relapse effects by engaging astrocyte-mediated synaptogenesis.N = 22, Experiments 2–3: N = 63, Experiment 4: N = 20) were purchased from Charles River Laboratories  and were single-housed upon arrival in a temperature- and humidity-controlled vivarium with standard rat chow  and water available ad libitum. Rats were maintained on a 12 hr reverse light/dark cycle (lights off at 6 a.m.). All animal use protocols were approved by the Institutional Animal Care and Use Committee of the Medical University of South Carolina and were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals . At the time of surgery, all rats weighed 275–300 g. On the day of surgery, rats were anesthetized with an intraperitoneal (i.p.) ketamine (66 mg/kg) and xylazine (1.33 mg/kg) injection, and received ketorolac  for analgesia. A silastic catheter was implanted in the right jugular vein, attached to a cannula, which exited the animal’s back. Rats in Experiment 1 received a bilateral intra-PrL cortical infusion of AAV5.GfaABC1D::tdTomato ; co-ordinates- AP: +2.8, ML: +/−0.6, DV: −3.8 mm from bregma). Rats in Experiment 2 received a bilateral intra-PrL cortical microinjection of AAV5.GfaABC1D::Lck-EGFP  to label the fine membrane processes of astrocytes [CAG.Flex::Ruby2sm-FLAG (Titer: 5 × 1012 vg/mL) as well as intra-NAcore  injection of rgAAV.hSyn::Cre-WPRE.hGH  to specifically target PrL cortical-NAcore neurons for dendritic spine analyses [GfaABC1D::Lck-EGFP (titer: 1 × 1013 vg/mL) or AAV5. GfaABC1D::shTSP2-GFP. Injections (0.75 µL/hemisphere) were performed over a period of 5 min (0.15 µL/min) using a Nanoject II  and injectors were left in place for 5 min to facilitate viral diffusion from the injection site. Apart from the TSP2 RNAi construct, a gift from the Dong Lab [GfaABC1D::Lck-EGFP construct was purchased as a plasmid from Addgene, then packaged to produce viral particles at the University of North Carolina Gene Therapy Center viral vector core . Rats were allowed at least 5 days of post-operative recovery during which catheters were flushed daily with taurolidine citrate solution  and food and water were available ad libitum. For each experiment, ≥2 independent cohorts were used. Male Sprague–Dawley rats  and dissolved in sterile saline at 100 mg/mL.w/v NaCl) 30 min before each subsequent session, and all animals injected with N-acetylcysteine received 13 days of extinction training (Experiments 2–4). All YS animals received vehicle injections during extinction. This dose of NAC has previously been shown to reduce extinction responding on the active lever and provide an enduring reduction in cued and primed heroin seeking using the same heroin SA protocol [All experiments were performed between the hours of 8 a.m. and 6 p.m. All SA experiments were performed in standard MedPC operant chambers equipped with two retractable levers, a tone generator, and a cue light . The beginning of each session was signaled by illumination of the house light and lever availability. Rats were trained for 14 days (3 h/day) to lever-press for heroin on a fixed ratio 1 (FR1) schedule of reinforcement, whereby each individual lever press on the active lever (ALP) resulted in the presentation of cue delivery and infusion of heroin followed by a 20 s timeout whereby ALP elicited no drug delivery. On days 1–2, active ALP resulted in a light and tone cue delivery followed by a 100 μg/50 μL i.v. infusion of heroin hydrochloride dissolved in sterile saline. On days 3–4, rats received a 50 μg/50 μL infusion followed by a 20 s. timeout period, and on days 5–14, rats received a 25 μg/50 μL infusion. Yoked-saline (YS) control animals were paired to a randomly selected heroin SA animal, receiving non-contingent saline infusions in a temporally paired manner. SA criteria were set at ≥1 infusion per session on days 1–4 whereas animals were required to receive ≥10 infusions per session on days 5–14. Following SA, all rats entered extinction whereby ALP resulted in no cue or drug delivery. All rats received 12–13 days of extinction training (3 h/day). Beginning on day 4, a subset of animals received N-acetylcysteine  or vehicle . Thick sections (∼220 μm) containing the PrL region were prepared using a vibrating microtome  and maintained in oxygenated aCSF in a dish in a 32 °C water bath until recording. During recording, sections were perfused at a flow-rate of ~2 mL/min with oxygenated aCSF at 33 °C. The prelimbic layer V/VI was identified initially using a 4× objective ), and later was magnified for patching with a 40 × (0.8 NA) water-immersion lens fitted to an upright microscope  fitted with infrared differential interference contrast optics.Twenty-four hours after the last extinction session, rats were anesthetized with isoflurane, decapitated, and their brains quickly removed and placed in an ice-cold and oxygenated artificial CSF (aCSF), slicing solution containing (in mM): 126 NaCl, 2.5 KCl, 1.2 MgClGfaABC1D::tdTomato) that were excited by wide-field illumination . Light power and duration were controlled via the LED driver  by external voltage modulation. Successful patching onto astrocytes was confirmed by electrophysiological features including their hyperpolarized resting membrane potential , their linear current–voltage relationship, their inability to generate APs, and their low membrane resistance . Recordings were taken with borosilicate glass pipettes (4 to 5 MΩ) containing the following internal solution: 115 mM K-gluconate, 6 mM KCl, 5 mM glucose, 7.8 mM Na-phosphocreatine, 4 mM Mg-ATP (adenosine triphosphate), 0.4 mM Na-GTP (guanosine triphosphate) , low-pass filtered at 2 kHz, and digitized at 10 kHz). Data were collected using AxoGraph X and stored for offline analysis. For the recording of the threo-beta-benzyloxyaspartate (TBOA) (non-selective blocker of excitatory amino acid transporters)- and BaCl2 (K+ channel antagonist)-sensitive currents, the extracellular solution contained antagonists of n-methyl-D-aspartate, (NMDA) receptors , ⍺-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptors , and γ-aminobutyric acid type A (GABAA) receptors . Whole-cell recordings were taken from astrocytes in PFC layers II/III and V by an experimenter blinded to treatment condition. Cell bodies of astrocytes were visualized using astrocyte-specific AAVs (AAV5.2 (200 µM)-sensitive currents were evoked by single-pulse stimulation every 20 s using focal electrical stimulation with a bipolar tungsten electrode (FHC) positioned immediately medial (within ~100 μm) to the recorded astrocyte. The protocol was repeated 2 times per stimulation intensity and then averaged and analyzed. Access resistance was monitored (<20 MΩ), and recordings with an access resistance changing more than 20% between the beginning and the end of the recording were discarded. Resting membrane potential (RMP) and input resistances (Rm) were monitored for analysis of the electrophysiological properties of astrocytes. Astrocytes were held at −80 mV, and TBOA (100 µM)- and BaClw/v paraformaldehyde (PFA) in 0.1 M PB. Brains were extracted and post-fixed in PFA for 24 h. Sections (80 µm) containing the PrL cortex and NAcore were collected in 0.1 M PBS containing 0.02% sodium azide with a vibrating microtome (Leica), and stored at 4 °C until processing.Twenty-four hours after the final extinction session, rats were transcardially perfused with 150 mL of 0.1 M phosphate buffer (PB), followed by 200 mL of 4% v/v) TritonX-100 (PBST) containing 2% (v/v) normal goat serum (NGS). Sections were then incubated overnight with gentle agitation at 4 °C with the following primary antibodies: Experiment 2—chicken anti-GFP  + mouse anti-GluA2  or rabbit anti-Synapsin I , and in a separate run, rabbit anti-GFAP ; Experiment 3—mouse anti-FLAG  and rabbit anti-GluA1 ; Experiment 4—rabbit anti-NeuN . Sections were then washed 3 × 5 min with PBST, and were incubated in species-specific secondary antibodies  conjugated to Alexa Fluor® 488 , 594 , or 647  diluted in PBST with 2% NGS for 5 h at room temperature (~20–25 °C) with gentle agitation. Sections were washed 3 × 5 min in PBST, then mounted on Superfrost plus slides with Prolong Gold antifade . Slides were stored at 4 °C until imaging (<2 months).Immunohistochemistry (IHC) was performed as previously described ,14,31. B® 488-mediated Lck-EGFP detection was carried out using an Argon 488 nm laser line, Alexa® 594-mediated FLAG detection was carried out using an OPSL 552 nm laser, and Alexa® 647-mediated GluA2, GFAP, and Synapsin I detection was carried out using a Diode 638 nm laser. In Experiment 2a, fields of GFAP + cells were imaged in layers II/III and V of the PrL cortex at a 4096 × 4096 frame size, 0.1 mm Z-step size, and 1× digital zoom, with a line average of 4 using a 63× oil-immersion objective (1.4 NA). In Experiment 2b, Lck-EGFP-expressing astrocytes that were visually isolated from neighboring astrocytes in layers II/III and V as well as GluA2/Synapsin I puncta were sampled using a 63× oil-immersion objective (1.4 N.A.) at a 2048 × 2048 frame size, 0.3 mm Z-step size, and 1.5× digital zoom, with a line average of 4. Care was taken to ensure that the majority of each astrocyte in the Z-plane was imaged. In Experiment 3, apical tuft dendrites of layer V PrL-NAcore neurons were imaged as previously described [For all high-resolution confocal microscopy experiments, a Leica SP8 confocal microscope  equipped with HyD detectors for enhanced sensitivity was used. Laser power, gain, and pinhole size (≤1 Airy Unit) were empirically determined to obtain optimal saturation values and subsequent deconvolution results. Laser power and gain values were then held relatively constant for the remainder of the experiment. For all experiments, Alexaescribed . BrieflyAll image analyses were performed by an investigator blind to experimental groups. Following acquisition, confocal Z-stacks were deconvolved using Huygens software v.2  and then exported to Bitplane Imaris v. 9.1  for digital rendering and analysis. Complexity of GFAP arbors were analyzed as previously described . BrieflyThe whole-cell astrocyte synaptic contact assay was performed as described previously ,14, withH) of each segment and the dendritic spine density (spines/µm). We also binned the spine head diameters (0.2 µm bins) to generate a frequency distribution. For GluA1 co-registry analyses, a 3D rendering was then used to isolate the dendrite segment (shaft + spines), and GluA1 signal above background contained within the dendrite that was isolated for analysis. The colocalization module of Imaris was then used to generate a channel containing GluA1 and FLAG colocalization, and the ‘filament analysis’ MATLAB Xtension was used to split the channel into FLAG-GluA1 colocalization in dendritic subcompartments including the dendritic spine head and dendritic shaft for differential investigation of colocalization in respective subcompartments. Output variables included the percent of GluA1 volume normalized to the dendrite volume, the total volume of spine-head-specific GluA1 normalized to the dendrite volume, and the total volume of dendritic shaft GluA1 normalized to the dendrite volume, as well as signal intensity controls. Data are expressed as animal averages, collapsing each of the 3–5 dendrites sampled per animal into an average value of spine dH and density per animal.Dendritic spine morphometric feature and GluA1 co-registry were analyzed as previously described . Brieflyq values are reported. If data were sampled from a normal distribution, but between-group standard deviations were not equal, a Brown–Forsythe-corrected one-way ANOVA was performed followed by Dunnett’s T3 test when a significant effect of treatment was revealed. When three groups were compared across a repeated measure, a repeated-measure one-way ANOVA was applied followed by Tukey’s multiple comparison test comparing the mean of the repeated measure between groups. Dendritic spine frequency distribution data and dendritic spine GluA1 colocalization data were analyzed with a two-way repeated-measure ANOVA followed by Tukey’s multiple comparison test when a significant interaction was revealed. GluA1 immunoreactivity across dendritic spine head size was evaluated using linear regression, wherein all figures contain linear fits derived from the analysis. Unless otherwise stated, each data point represents an animal average derived from multiple images or SA/extinction sessions. Statistical outliers were detected according to the ROUT method (Q = 1%) and these data were removed from analyses as specified in the results. All data are expressed as the mean ± SEM, and significance was set at p < 0.05. A priori sample size and power estimates were derived from previously published literature [All statistical analyses were performed with SPSS statistical software  or Graphpad Prism software . Evoked GLT-1-dependent current from whole-cell astrocyte recordings was modeled with non-linear regression techniques, and a linear discriminant analysis with bootstrapping procedure was performed to determine if evoked GLT-dependent current was predictive of initial drug treatment. A principal components analysis was performed to produce a lower-dimensional data set before a clustering analysis was performed for all cells recorded. Self-administration and extinction data across subsequent days were compared using a mixed-model ANOVA. When analyzing three groups across a single dependent variable, a one-way ANOVA was performed followed by Tukey’s multiple comparison test if a significant effect of treatment was revealed. Normality of data within each group were analyzed using Shapiro–Wilk test. If one or more groups for each variable analyzed did not pass the Shapiro–Wilk test, a non-parametric Kruskal–Wallis test was used. In this instance, post hoc tests controlled for the false discovery rate (FDR) using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli if a significant effect was revealed, in which case FDR-corrected terature ,31.+ or glutamate current was evaluated. The current attributed to glutamate uptake was blocked with the excitatory amino transporter inhibitor TBOA, whereas K+ conductance was blocked with BaCl2, a K+ channel antagonist. Of the 22 initial animals in Experiment 1, 1 animal did not finish SA due to catheter failure. N = 11)- and heroin (N = 10)-treated animals. Comparing heroin and YS active lever presses across sessions, a mixed-model ANOVA reveals a significant drug-by-time interaction  = 3.002 p < 0.001). From sessions 9–14, heroin SA animals pressed the active lever significantly more than YS controls  = 11.30, p = 0.003). In Experiment 1, we sought to evaluate the effects of heroin SA and extinction upon the physiological properties of PrL astrocytes using a strategy where an electrical-stimulation-induced inward current attributed to either the KN = 10) and YS-treated animals (N = 11) were euthanized 24 h after the final extinction session and evoked K+ and GLT−1-dependent currents were recorded from layer II/III and V astrocytes . + current in PrL astrocytes following a single stimulation in saline- and heroin-treated rats. No difference was found for the evoked K+ current . Next, to determine the degree to which drug treatment is predictive of the evoked GLT-1 response, the data were first bootstrapped (sample n = 1000) before a linear discriminant analysis was performed, where we show that the treatment condition can be predicted with 85.4% accuracy . Obtained discriminant scores are displayed in To examine functional adaptations in cortical astrocytes following heroin SA and extinction, heroin-treated animals ( current D. Margin current E. To furN = 31) and YS (N = 20) rats used for Experiments 2–3. Of the 63 initial rats in Experiments 2–3, 12 animals did not finish SA due to catheter failure. When comparing heroin and YS active lever presses across SA sessions, a two-way repeated-measures ANOVA revealed a significant group-by-time interaction  = 5.112, p < 0.001). Heroin SA animals pressed the active lever significantly more than YS controls in sessions 9–14  = 10.168, p = 0.002). There was no difference between groups (that were subsequently divided by treatment during extinction) in the average number of heroin infusions earned over the last 3 days of SA , indicating that the observed effects of NAC upon extinction were not dependent upon differences in initial heroin intake during SA. N = 20), and heroin SA rats injected with vehicle (N = 15) or NAC (N = 16) during the last 10 days of extinction. p < 0.0001). Tukey’s multiple comparison tests revealed that heroin SA animals showed significantly more lever pressing during the last 3 days of extinction than YS rats (p < 0.0001) and that NAC significantly decreased lever pressing to YS values (p = 0.019).GfaABC1D::Lck-EGFP) to evaluate GFAP arbor complexity following heroin SA, extinction, and NAC treatment. p = 0.30, p = 0.0005) and Heroin + NAC  groups were non-normally distributed. Accordingly, a Kruskal–Wallis non-parametric ANOVA was used, which indicated a significant difference between groups . The two-stage linear step-up procedure revealed that heroin increased total GFAP filament length per image when compared to YS (q = 0.0007), which was normalized by NAC treatment during extinction (q = 0.004). We next analyzed the average GFAP filament diameter for each cell per Z-series data set (p = 0.04). As such, these data were analyzed using a Brown–Forsythe-corrected ANOVA which revealed there was no significant effect of treatment  = 0.626, p = 0.54). We next analyzed the average number of branch points of each cell, within each Z-series data set . The two-stage linear step-up procedure revealed that heroin increased the average number of branch points within GFAP filament arbors compared to YS (q = 0.0002) which was normalized by NAC treatment during extinction (q = 0.003). We also performed a 3D Sholl analysis for each cell within each image using 1 µm 3D concentric sphere radii (which were then compiled into 5 µm bins) and analyzed the average number of intersections as a function of distance from the center of each cell (p < 0.0001). Tukey’s multiple comparison test revealed that heroin increased the average number of Sholl intersections compared to YS (p = 0.0002), which was normalized by NAC treatment during extinction (p < 0.0001). In a subsequent analysis, we focused on the first 15 µm 3D sphere radius from the centroid of each cell to investigate the primary GFAP+ branches (p = 0.0004). Tukey’s multiple comparison test revealed that heroin increased the number of Sholl intersections in this region when compared to YS (p = 0.003), which was normalized by NAC treatment during extinction (p = 0.0007). Here, we used an astrocyte-specific membrane-targeted EGFP reporter virus to label the astrocyte plasma membrane (AAV5-data set D. One Z-data set E. One Z-ach cell F. A repebranches G. In theN = 10), Heroin + Vehicle (N = 9), and Heroin + NAC (N = 10), to examine the association of astrocytes with synaptic markers following heroin SA, extinction, and NAC treatment. In the first set, the astrocyte structure was visualized with viral-vector-mediated membrane-targeted EGFP expression achieved with the AAV5.GfaABC1D::Lck-EGFP viral vector and further amplified with GFP IHC whereas excitatory synapses were labeled with GluA2 IHC as described previously [We performed two separate astrocyte–synaptic contact analyses using PrL cortical sections collected from the three experimental groups, YS (eviously . The seceviously . We imagp < 0.01). The two-stage linear step-up procedure indicated that the Heroin + Vehicle group showed an increased co-registry of GluA2 and GFP compared to the YS group (p = 0.01), which was normalized by repeated NAC treatment during extinction ; Heroin + Vehicle: lack of available tissue (N = 1) or insufficient Lck-EGFP virus expression (N = 1); Heroin + NAC: lack of a sufficient number of analyzable cells after imaging (N = 1); and technical issues related to IHC (N = 1). A one-way ANOVA revealed a significant main effect of treatment  = 6.51; p = 0.006; p = 0.012), which was normalized by NAC treatment during extinction (p = 0.012). Akin to analyses performed for GluA2, we also analyzed the number of Synapsin I voxels in a subset of images from each animal (p = 0.04) were non-normally distributed. A Kruskal–Wallis test indicated no difference between groups . There was no difference between groups in astrocyte complexity as measured by the ratio of the surface area to volume for cells used in either the GluA2 or Synapsin I data sets , Heroin + Vehicle (N = 6), and Heroin + NAC (N = 6) animals after extinction. H) with warmer colors indicating larger values. We first analyzed the average dendritic spine dH across the three treatment groups. A one-way ANOVA revealed a significant effect of heroin treatment  = 20.70, p < 0.0001, H (p < 0.0001), which was not altered by NAC treatment during extinction. Heroin + NAC treatment likewise increased dH relative to saline-treated animals (p = 0.0007). Next, we investigated whether this effect was specific to smaller or larger spines. To do so, we binned the spine dH (0.2 µm bins) and generated a frequency distribution. A two-way repeated-measures ANOVA revealed a significant treatment-by-bin interaction  = 8.144, p < 0.0001, p < 0.0001; 0.2–0.4—p < 0.05), but increased the proportion of spines with a larger dH (0.4–0.6—p < 0.01) compared to YS rats. Interestingly, the proportion of spines with a dH between 0.6–0.8 was increased in the Heroin SA + Vehicle group (p = 0.008), but there was no effect of NAC on this specific bin compared to the YS treatment group (p = 0.17). We next analyzed the average dendritic spine density in all three treatment groups from the same dendrite segments. A one-way ANOVA revealed a significant effect of treatment  = 13.46, p = 0.0002, p = 0.0003), which was prevented by NAC treatment during extinction (p = 0.002). To determine which spine head diameter bins accounted for the altered spine density, we performed a similar analysis as in p < 0.0001, p < 0.0001), regardless of NAC treatment (p = 0.0003), decreased the density of spines with a head diameter less than 0.2 µm. However, the Heroin + Vehicle group showed decreased spines in the 0.2–0.4 µm bin compared to YS (p < 0.0001), which was normalized in the Heroin + NAC group (p < 0.0001 compared to Heroin + Vehicle). Interestingly, the density of larger spines with head diameters between 0.4–0.6 µm were unaffected in the Heroin + Vehicle group compared to YS (p = 0.194), yet Heroin + NAC increased the density of these spines compared to the YS (p = 0.0125) and Heroin + Vehicle (p = 0.0002) groups. These effects on dendritic spine morphometric properties were likely not due to inherent differences in the FLAG signal intensity within the spine heads as there was no difference between groups in the average FLAG intensity within the spine head region when normalized to saline controls . Tukey’s multiple comparison test indicated that the Heroin + Vehicle (p = 0.02) and Heroin + NAC (p = 0.0009) groups displayed increased GluA1 immunoreactivity compared to saline controls  were associated with altered GluA1 immunoreactivity in the discrete dendritic subcompartments of the PrL-NAcore apical tuft dendrites described above. When analyzing the total volume of GluA1 immunoreactivity in the dendritic spine heads and shaft combined, relative to the total dendrite volume, a one-way ANOVA revealed an omnibus significant effect of NAC treatment upon GluA1 immunoreactivity  = 10.56, controls A. In thep = 0.0005). Dunnett’s T3 multiple comparison test revealed that the Heroin + Vehicle (p = 0.03) and Heroin + NAC (p = 0.004) groups showed increased spine-head-specific GluA1 signal accumulation when compared to the Saline + Vehicle group  = 20.22, le group B. Howevele group C.p = 0.004). Tukey’s multiple comparison test indicated that the Heroin + Vehicle (p = 0.033) and Heroin + NAC (p = 0.005) groups showed increased dendritic-shaft-specific GluA1 immunoreactivity compared to the Saline + Vehicle group  = 7.73, le group C. The Hele group D. This iGfaABC1D::Lck-EGFP  or AAV5.GfaABC1D::shTSP2-GFP , an shRNA targeted to TSP-2 [p = 0.013) and the delivery of shTSP-2  = 5.005, p = 0.037) significantly reduced active lever pressing during the last three days of extinction. Though NAC+ shTSP-2 treatment did result in a marginal decrease in active lever pressing during the last three days of extinction, the effect was not statistically significant. Similarly, both NAC  = 6.155, p = 0.02) and the delivery of shTSP-2  = 4.423, p = 0.04) significantly blunted cued-relapse to heroin seeking. Similar to findings from extinction data, NAC + shTSP-2 treatment resulted in a marginal decrease in cued-relapse active lever pressing, though the effect was not statistically significant compared with either the Lck+ NAC or shTSP2 + vehicle treatment groups . As It is possible that the heroin-induced decrease in spine density could be linked to dysfunctional gliotransmission following extinction, and that NAC’s ability to drive spinogenesis could be linked to the normalization of such dysfunction. For example, repeated morphine administration in mice downregulates TSP expression in the cortex 24 h after the final injection, as well as in cultured astrocytes following repeated morphine treatment . One mecGiven that one well-documented mechanism by which NAC counteracts oxidative stress is increased glutathione synthesis , it is pNAC has previously been demonstrated to regulate glutamate homeostasis, which has been consistently shown to be disrupted following drug self-administration ,16,17,24The most obvious limitation to the present set of experiments is the omission of female rodents. These series of studies were initially planned as a follow-up to existing literature surrounding astrocyte–neuron interactions in the NAcore . BecauseAnother limitation of the present findings is that they are largely correlative in nature, and our present experimental manipulations do not allow us to infer causality between heroin adaptations to neurons and astrocytes, cued-relapse to heroin, and the modulatory role that NAC plays in this process. Future experiments will involve targeted manipulations of these specific features in an attempt to establish causal relationships between the relationships discussed.While we show that the evoked astrocytic GLT-1-dependent current can be a predictive measurement of prior heroin exposure, it is unclear as to the functional ramifications this effect has owing to the relatively marginal differences observed between the evoked GLT-1-dependent current between YS- and heroin-treated animals. Moreover, as discussed above, it is unclear if any such alterations play a role in cued-relapse to heroin.Another limitation of the present experiments was the lack of YS control animals treated with NAC. In the current study, we chose to omit YS animals injected with NAC for several reasons. Most importantly, NAC has been shown to have no effect on NAcore glutamate concentrations or cystine uptake through Xc- in control animals . MoreoveCanonically, GFAP is thought to only be present in the major branches and endfeet of astrocytes and not to be present in all non-reactive astrocytes in adult healthy CNS tissue . HoweverGfaABC1D::Lck-EGFP virus as a negative control as opposed to an off-target shRNA construct which is also a limitation of the present study.Finally, in our shTSP2 experiments, we utilized the construct provided by the Dong Lab. Validation of TSP2 knockdown was performed by the Dong Lab in HEK293 cells and reduced cocaine-mediated synaptogenesis in vivo. . QuantitIn conclusion, our data indicate that heroin SA and extinction elicit hypertrophic complexity and synapse association of astrocytes in the PrL cortex that are associated with altered dendritic spine morphological features in PrL-NAcore neurons. Our data fill a much-needed gap in the field by focusing on the upstream portion of the canonical relapse circuit to accompany what has been observed in the NAcore. Moreover, these findings point to an additional node of this circuitry whereby NAC can modify heroin-induced adaptations in the PrL cortex in addition to its well-defined modification of NAcore synaptic pathology. Ultimately, astrocytic and neuronal adaptations in the PrL cortex may be a target for informing future drug discovery for relapse prevention pharmacotherapies.


ROS1-rearranged NSCLC that responded to crizotinib after tumor progression due to MET polysomy during entrectinib treatment. This case suggests that crizotinib is an effective option for patients with MET polysomy, even after disease progression on entrectinib.Resistance to ROS1 tyrosine kinase inhibitors is inevitable, but it has been unclear whether crizotinib might be effective after the development of entrectinib resistance. We here present a case of  ROS1 rearrangement account for approximately 1% to 2% of NSCLC cases,ROS1-rearranged NSCLC. Nevertheless, resistance to entrectinib inevitably develops. The mechanisms of such resistance remain unclear in most cases, and it is not known whether crizotinib is effective after the development of entrectinib resistance. We now report a case of resistance to entrectinib that was characterized by next-generation sequencing (NGS) analysis and overcome by crizotinib.Tumors with EGFR mutation nor ALK translocation. She received nivolumab as a third-line treatment and achieved a partial response. After 25 months, a CT scan identified tumor progression in the right lung. S-1 was then administered as a fourth-line treatment for two cycles, after which disease progression was again apparent. A bronchoscopic biopsy from the upper lobe of the right lung was again performed, and the tissue was subjected to NGS (Archer FusionPlex CTL panel), which revealed the presence of an SLC34A2-ROS1 fusion gene.A 64-year-old woman who had never smoked presented with dyspnea. A computed tomography (CT) scan revealed a 29-mm nodule in the right upper lobe of the lung, enlargement of the right hilar lymph node and bilateral supraclavicular lymph nodes, and pleural effusion. She was diagnosed with having stage IV lung adenocarcinoma accompanied by malignant pleural effusion. She received first-line treatment with carboplatin plus pemetrexed for four cycles, resulting in a confirmed partial response as the best response . After 1The patient received entrectinib (600 mg/d) as a fifth-line treatment, and she achieved a partial response (−30%) in the upper lobe of the right lung at 3 months . The entMET gene copy number of 3 in both specimens, but neither secondary mutations in the ROS1 kinase domain nor activation of bypass signaling pathways was detected. Fluorescence in situ hybridization (FISH) analysis of MET amplification revealed MET polysomy with probes for MET and CEP7. The pretreatment specimen revealed 5.8 signals per cell for MET and indicated a MET-to-CEP7 ratio of 1.1, whereas the posttreatment specimen revealed 6.1 signals per cell for MET and indicated a MET-to-CEP7 ratio of 1.1  or low (>1.8 to <2.2), intermediate (>2.2 to <5.0), or high (>5.0) amplification.MET and CEP7 copy numbers. The analysis revealed MET polysomy on the basis of a copy number gain  and low ratio of MET to CEP7 (1.1). Both entrectinib and crizotinib are tyrosine kinase inhibitors for ALK and ROS1. In contrast to entrectinib, however, crizotinib does not inhibit TRK but is a potent inhibitor of MET. A patient with NSCLC having a MET copy number gain without a high MET-to-CEP7 ratio was previously reported to experience a rapid response to crizotinib.MET as determined by FISH, including those with MET polysomy, had the best objective response rate (32%) and median progression-free survival (3.2 mo).MET polysomy in these previous studies,,MET polysomy benefit from crizotinib. MET polysomy might therefore have the potential to be an oncogenic driver that is targetable by crizotinib.In the present case, although the MET polysomy before entrectinib, she had an initial response to entrectinib treatment. It has been proposed that intratumoral heterogeneity is associated with a differential drug response.ROS1 rearrangement in the present patient might have been inhibited by entrectinib, a small subpopulation of cells also harboring MET polysomy might have survived during continuous entrectinib treatment and become the dominant population in the entrectinib-resistant tumor.Although the current patient had MET amplification or polysomy, even after disease progression during entrectinib treatment. Further study is warranted to understand the mechanisms of entrectinib resistance in ROS1-rearranged NSCLC and the efficacy of crizotinib in NSCLC with MET polysomy.Our report suggests that crizotinib is an effective option for patients with Toshiaki Takakura: Writing—original draft, Investigation, Data curation.Hiroaki Kanemura, Kazuko Sakai, Kazuto Nishio: Investigation, Data curation.Hidetoshi Hayashi: Supervision, Writing—review and editing, Conceptualization.