Differential responses to drought of two genotypes were obtained: more pronounced stomatal closure, ABA biosynthesis and proline accumulation in WT compared to the mutant were compensated by dry weight accumulation accompanied by transient redox disbalance in flacca. Fourier-transform infrared (FTIR) spectra analysis of isolated cell wall material and morphological parameter measurements on tomato leaves indicated changes in dry weight accumulation and carbon re-allocation to cell wall constituents in flacca, but not in WT. A higher proportion of cellulose, pectin and lignin in isolated cell walls from flacca leaves further increased with repeated drought cycles. Different ABA-dependent stomatal closure between drought cycles implies that acquisition of stomatal sensitivity may be a part of stress memory mechanism developed under given conditions. The regulatory role of ABA in the cell wall restructuring and growth regulation under low leaf potential was discussed with emphasis on the beneficial effects of drought priming in developing differential defense strategies against drought.Two tomato genotypes with constitutively different ABA level,  Water scarcity has become one of the greatest problems for agricultural production, with rapidly increasing instances in many areas of the world. Drought is identified as a major threat to crop production causing billions in annual economic losses, mainly from the agricultural sector ,2. The iStress imprinting (or priming), which positively affects plant survival under repetitive stresses, is widely accepted as an alternative agriculture measure to prepare plants to withstand drought episodes accompanied with high insolation and heat waves ,21. PrevLycopersicon esculentum Mill.) are one of the most important and widely grown vegetables in the world, especially in the Mediterranean region [Tomatoes  is a key molecule that underpins the regulatory mechanisms of a plant’s response to drought, such as the regulation of the turgor pressure of guard cells and stomatal closure, maintenance of water balance, osmotic stress tolerance and activity of antioxidative enzymes ,34,35. IRecent studies have demonstrated that ABA may contribute to increased drought tolerance related to drought memory effect ,40. The NCEDs) [Elevated ABA content under drought is predominantly the result of de novo biosynthesis of genes included in ABA metabolism, such as 9-cis-epoxycarotenoid dioxygenase (NCEDs) ,43. MoreNCEDs)  and/or ANCEDs) .flacca, which, compared to wild type, accumulates a lower content of ABA [flacca, due to the deletion of six base pairs in a molybdenumcofactor (Mo-Co) sulfurase, sulphuration of Mo-Co in AO is inhibited and, therefore, their oxidative activity is suppressed [In this study, we used one of the tomato (Ailsa Craig cv.) ABA-deficient mutants, t of ABA .The lastppressed ,46. Thesppressed ,48,49,50ppressed ,48,49.flacca mutant exhibited constitutively higher levels of soluble sugars and free amino acids (AAs) compared with its parent line Ailsa Craig cv. [In this work, we compare a plant’s response to repeated drought cycles (watered/drought-induced/re-watered plant status) during the vegetative development of two tomato genotypes, differing in the constitutive leaf ABA level, with the final aim to explore the ABA role in short term stress memory and acclimative mechanisms to water deficit. Recently, we reported that the same raig cv. . Here, wflacca, compared to its parent line (p = 0.000000). Water withdrawal to the same soil water content (SWC) during 6 days in the 1st drought cycle induced a 65% and 15% increase in ABA concentration, flacca, respectively. In all three drought periods, ABA accumulation in flacca was similar to the values measured in turgid WT leaves. While the ABA content in WT bounced back after three days of rehydration to levels found prior to water scarcity in all three applied cycles; the same was obtained only in the 2nd and 3rd cycles in flacca.Leaf ABA content in both genotypes did not vary throughout the growth period under well watered conditions, with 20–30% less ABA concentration in the ABA-deficient mutant of Ailsa Craig, flacca mutant showed significantly higher stomatal conductance in comparison to WT plants throughout the whole period, with the least measured difference found in the 2nd cycle (p = 0.000001). Stomatal conductance in WT drought-stressed plants varied in inverse proportion to the ABA accumulation, with the strongest response in the 3rd cycle. However, a statistically significant, but much smaller decrease compared to WT in the stomatal conductance, was also observed in flacca in the 2nd and 3rd drought episodes  under well watered conditions did not differ between genotypes; however, in the 1st drought this decreased to a larger extent in WT than in rd cycle .flacca more than 7 times compared to turgid leaves. An initial proline concentration was reset upon re-watering, in the 1st cycle in both genotypes and in the 2nd cycle only for WT. In the other two drought episodes, proline content did not change in comparison to respective controls  the control plants, which were continuously irrigated, (2) the plants that experienced one drought stress followed by 15 days period of re-watering, and (3) the plants that experienced three drought cycles.flacca increased; however, the effect was more pronounced in the mutant , 1635 cm−1 , 1420 cm−1 , 1240 cm−1 (C–O stretching) are characteristic for pectin, while the band at 1517 cm−1 is characteristic for lignin. The bands at 1147 cm−1 (O–C–O asymmetric stretching glycosidic link) and 1071 cm−1  are typical for xyloglucan, and the band at 896 cm−1 is related to cellulose, hemicellulose and pectin.Overlaid WT and flacca mutant leaves of tomato obtained from the FTIR spectroscopy was analyzed using principal component analyses (PCA). PCA was applied on a spectral range (800–1800 cm−1) where the first principal component absorbs 72% of the total variance (PC1), while the second component (PC2) absorbs 25%  are negatively correlated with PC2. According to scores and loadings for PC1, bands characteristic for cellulose, pectin and lignin are the most pronounced in flacca R3 and less in flacca control plants. The results obtained demonstrate that plants after three drought episodes have elevated contents of cellulose, pectin and lignin compared to flacca plants after prolonged recovery and to corresponding controls. According to FTIR spectra analyses, the content of pectin in WT plants remains unchanged between control plants and plants with different drought history and recovery periods. However, in flacca plants, drought induced pectin accumulation, with the most prominent increase in plants after three drought cycles. On the other hand, in WT plants, the relative intensity of bands characteristic for cellulose and xyloglucan was the highest in plants after prolonged recovery. Additionally, the content of these polysaccharides was elevated in plants after three drought episodes compared to control plants. Contrarily, in flacca plants, three drought cycles caused the greatest accumulation of cellulose, hemicellulose and xyloglucan, while in the prolonged recovery a slight increase in their accumulation was observed in comparison to control plants.The effect of prolonged recovery, and/or three drought cycles on the content of specific components of the cell wall in the WT and orbs 25% . PC1 negflacca is reduced by around 25% and exhibits a phenotype  that is similar to other ABA-deficient tomato mutants described in the literature [ABA plays a pivotal role in regulating stomatal conductance and transpiration rate and, finally, in developing a plant’s drought tolerance . In our terature ,57,58,59terature ,32,60.flacca a similar trend was observed only in the 2nd drought episode. We suppose that some form of ABA conjugates could dominate at that particular stage as a backup for potential upcoming drought [Despite differences in constitutive ABA content, the exposure of plants to water deficit in all three drought cycles provoked ABA accumulation in both genotypes, though to a higher extent in WT . While t drought .The results obtained could be explained as an ABA-induced “after effect”, as it could control stomatal conductance and transpiration rate in the early recovery phase . This ABNCED1 gene under water deprivation is in line with the observed ABA accumulation in tomato leaves . Under water deficit, though, stomata partly closed even in flacca, and transpiration rates were maintained higher in all three cycles in comparison to WT (two to five times higher gs in flacca). However, flacca’s stomatal aperture was also the most responsive to water deficit in the 3rd cycle in spite of differences in gs between genotypes throughout the experiment , while stomata almost closed in the 3rd cycle , we suppose that the drought-induced accumulation of dry biomass obtained in  and SLA . Such moThe stimulation of photosynthesis following drought and recovery has also been obtained in other species ,101. AltDrought-induced cell wall remodeling includes changes in architecture, accumulation, and cross-linking of cellulose and hemicelluloses–xyloglucan polymers , thus, cflacca leaves were observed in recovered plants after three drought cycles. Cellulose, hemicellulose in total and xyloglucan, as a part of the most dominant hemicellulose polysaccharides, were significantly elevated in recovered plants after three drought cycles, as well as lignin polymers. Likewise, the most prominent changes in the cellulose–hemicellulose complex in WT were observed in recovered plants after one drought, while in flacca, this was noticed after three drought episodes. Drought-induced cellulose and hemicellulose accumulation contribute to maintaining cell turgor pressure and cell wall mechanical strength and rigidity, which supports cell protection from water deprivation and permitting their continuous growth [Based on the extensive comparison analysis of FTIR spectra of cell walls isolated from both genotypes , we furts growth ,112.Increased lignin deposition and up-regulation of enzymes related to its biosynthesis and accumulation under drought conditions were also reported in numerous articles ,115,116.flacca leaves after three drought cycles. Nonetheless, one and/or three drought episodes in WT plants did not influence pectin content, and it remained unchanged. With respect to water stress, the amount of side chains of pectic polymers significantly increased in drought tolerant cultivars [Nicotiana sylvestris L. and H. annuus leaves, respectively [We also demonstrated the drought-induced biosynthesis of pectin, of which the content, as with other analyzed CW compounds, i.e., cellulose, hemicellulose and lignin, accumulated preferentially in ultivars . Interesectively ,119. A hflacca plants. The tightening and loosening of cell walls accompanied by changes in the cell wall composition are processes tightly related to cell growth and regulated by various stresses [The drought-induced cell wall thickening of water-conducting and supporting tissues  would costresses . Water sflacca after three drought cycles implies that the drought acclimation mechanism was driven through morphological changes, and that prior drought cycles poorly contribute to drought tolerance; rather it is the duration of re-watering periods that are more important.However, cell wall thickening presumably increasing with each subsequent drought cycle may create some kind of physiological memory and, consequently, plants’ higher drought tolerance. Taken together, the accumulation of the aforementioned cell wall components being the most evident in flacca mutant tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig) seeds were germinated in pots containing commercial substrate Klasman Potgrond H. Following the phase of four developed leaves, plants were transferred to larger pots (a depth of 24 cm). Plants were grown under controlled conditions with a light intensity of 250 μmol m−2 s−1, photoperiod 14/10 h (day/night), day/night temperature of 26/17 °C, and 50% relative humidity. Volumetric soil water content (SWC) was continuously maintained at 38 ± 2%. In the phase of 6 leaves, plants from both genotypes were transferred to a light intensity of 800 μmol m−2 s−1 with the same conditions of photoperiod, temperature, and humidity. After 4 days, plants (start) were divided into three groups. The first group was regularly irrigated during the whole experiment , the second was exposed to just one drought episode (D) and then optimally irrigated (R), while the third was subjected to three drought cycles (D) with recovery periods (R) of three days after each drought. Plants subjected to water deficit were not irrigated until reaching 11 ± 2% moisture in the substrate. Recovered plants from all drought cycles achieved SWC of 38 ± 2% immediately after the first day of the re-watering (Wild type (WT) and watering .For each treatment, four biological replicates of both genotypes were prepared. All of the samplings for biochemical measurements and measurements of stomatal conductance and relative water potential were performed using the fourth fully expanded leaf. Sampled leaves were immediately frozen in liquid nitrogen and stored at −80 °C for further analysis.s) was measured by porometer . Leaf water potential  was carried out with a Scholander-type pressure bomb . Fresh and dry weight of both, WT and flacca genotypes was determined upon all three drought episodes and after 3-days of recovery period, in treatment, as well as in control plants. Total leaf area (LA) was conducted by LI-3100 areameter , and specific leaf area was calculated using the equation: SLA = Leaf area/DW. All measurements were performed with four different plants per genotype and treatment.Volumetric soil water content (SWC) was measured using the Theta probe , and stomatal conductance (gDetermination of abscisic acid (ABA) content in the tomato leaves was performed as described in Živanović et al., 2020 . ABA conw/v) sulfosalicylic acid and centrifuged at 14,000× g for 10 min at 4 °C. The obtained supernatant was mixed with acidic ninhydrin and glacial acetic acid  and incubated for 60 min on 100 °C. The reaction mixture was placed on ice and extracted with toluene . The toluene fraction was used for determination of proline by measuring absorbance at 520 nm, with toluene as blank [In order to determine proline content, frozen leaf samples were homogenized in liquid nitrogen, extracted in 3% ; Phenomenex Ltd., Torrance, CA, USA) using the Shimadzu LC-20AB Prominence liquid chromatography . The elution gradient was formed with 20 mM potassium phosphate buffer, pH 6.5 (solvent A) and methanol (solvent B): 0–6 min, 30% solution B (isocratic conditions); 6.00–6.01 min, 55% solution B (isocratic conditions); 6.01–13.0 min, 55–30% solution B (linear gradient) at a flow rate of 0.8 mLmin−1. The fluorescence intensity of o-PDA-DHA derivative was measured at excitation and emission wavelengths of 360 and 430 nm using RF-10-AXL, fluorescence detector .The frozen leaf tissues were homogenized in 1.5% meta-phosphoric acid with 2 mM EDTA and centrifuged at 14,000× a et al. . Brieflya et al.  with somRNA was isolated by TRIzol™ Reagent  according to manufacturer’s protocol. RNA was quantified with a NanoDrop spectrophotometer , and its quality was checked by electrophoresis on 1.5% agarose gel. To remove traces of DNA contamination, RNA was treated with DNase I  at 37 °C for 10 min, according to the manufacturer’s protocol. Synthesis of cDNAs from 1 µg of total RNA was carried out in the reverse transcription reaction (RT), according to the manufacturer`s protocol RevertAid First Strand cDNA Synthesis Kit .NCED1 gene, which is involved in abscisic acid biosynthesis, was evaluated by quantitative real time PCR using SYBR green in 7500 Real Time PCR System . A total of 10 µL reaction mixture contained 1 μL of RT reaction product, appropriate forward and reverse primers and Maxima SYBR Green/Rox qPCR Master Mix . The forward primer 5′AGGCAACAGTGAAACTTCCATCAAG3′ and reverse primer 3′TCCATTAAAGAGGATATTACCGGGGAC5′ were used for NCED1 amplification (GenBankTMAccession No. Z97215). Thermal cycling conditions for qRT-PCR included: initial denaturation on 95 °C, then 40 cycles of denaturation (95 °C for 30 s), annealing (55 °C for 30 s) and extension (72 °C for 30 s). The absence of primer-dimer formation was checked in no-template controls. In an amplification reaction, the point in the cycle at which the target DNA has amplified enough so that its fluorescence reaches fixed threshold above the background fluorescence is called the Ct value of the sample. The expression level of the tested gene was normalized to the housekeeping gene actine and calculated relative to WT start control according to the ∆∆Ct method. The results are presented as log2 transformation of fold changes (log2FC). For each sample, qRT-PCR was performed in triplicate [Expression of iplicate .w/v) with shaking for 60 min at room temperature. The homogenate was centrifuged at 1000× g for 20 min at room temperature and pellet was washed two times with 80% methanol. The pellet was resuspended in 1 M NaCl with 0.5% Triton ×100 and centrifuged at 1000× g for 20 min at room temperature. The pellet was re-washed with distilled water, once with absolute methanol and twice with acetone. The purified cell wall obtained was dried and additionally powdered in Mixer Mill MM 400 (Retsch) and further used for structural analyses.The plant cell wall was isolated according to the procedure described in Simonović-Radosavljević et al. . The dri−1 with 200 scans and a spectral resolution of 4 cm−1. Baseline correction was performed using the trial Spectra Gryph software .The Fourier-transform infrared (FTIR) spectra of the extracted cell wall materials were recorded using a Perkin Elmer Spectrum Two equipped with the Universal ATR accessory. The spectrum of each powder sample was collected in the range of 4000–400 cms, ψ, morphological parameters, ABA, proline and ascorbate contents. Tukey post hoc test was used for specific comparisons among experimental groups and significance threshold value was set at p ≤ 0.05.Three-way ANOVA was used to reveal the effects of genotypes, number of drought cycles and water regimes and their interactions on gNCED1 gene expression. The experimental data processing was conducted using software package Statistica 8.0.Welch t-test was used to determine differences in −1 [X into score matrix (T), loading matrix (P) and residual matrix (E): T = TPT+ E. The similarity of the samples can be visualized by reducing the dimensionality of the space, i.e., their projection on a subspace defined with a smaller number of dimensions . If the first two principal components absorb a sufficient percentage of the total variations, then the samples can be projected onto a plane spanned by the vectors of these principal components, which facilitates the visual identification of pattern similarities.In order to investigate the similarities between the samples, principal component analyses (PCA) was performed on the FTIR spectra, in the region between 1800 and 800 cm−1 . PCA decNo preprocessing of data was required. The software Unscrambler X 10.4  was used for multivariate analysis.flacca mutant and its parental line Ailsa Craig cv.) developed different drought tolerant strategies depending on their constitutive ABA levels. A failure of the efficient stomatal closure mediated by drought-inducible ABA accompanied by lower leaf potential and transient oxidative stress are compensated by a double increase in the dry weight of flacca leaves in plants that experienced prolonged recovery. Drought induced more pronounced stomatal closure, ABA biosynthesis and proline accumulation in WT compared to the mutant, confirming the importance of stomata in water loss prevention in WT. The most efficient stomatal response observed in the 3rd drought cycle in WT, which did not correlate with ABA increase, implies that repeating drought cycles trigger the acquisition of stomatal sensitivity to a chemical and/or a hydraulic signal. On the other hand, the osmoprotective role of proline was not pertinent for developing drought stress memory, considering that the drought-induced accumulation of proline was dramatically decreased with repeating drought cycles.In this work, we demonstrated that analyzed tomato genotypes (flacca) and saturating, high growing light, modification of the cell wall occurred. Strengthening of the vascular tissue and cell wall capacity of water storage are traits favored by the enhancement of networking between cellulose, hemicellulose, pectin and hydroxyproline protein enrichment, and may present a target for future improvement of drought tolerance.We hypothesize that under certain conditions imposed by lower ABA content (


Large-scale intracellular signaling during developmental growth or in response to environmental alterations are largely orchestrated by chromatin within the cell nuclei. Chemical and conformational modifications of the chromatin architecture are critical steps in the regulation of differential gene expression and ultimately cell fate determination. Therefore, establishing chemical properties of the nucleus could provide key markers for phenotypic characterization of cellular processes on a scale of individual cells. Raman microscopy is a sensitive technique that is capable of probing single cell chemical composition—and sub-cellular regions—in a label-free optical manner. As such, it has great potential in both clinical and basic research. However, perceived limitations of Raman spectroscopy such as low signal intensity and the difficulty in linking alterations in vibrational signals directly with ensuing biological effects have hampered advances in the field. Here we use immune B lymphocyte development as a model to assess chromatin and transcriptional changes using confocal Raman microscopy in combination with microfluidic devices and correlative transcriptomics, thereby linking changes in chemical and structural properties to biological outcomes. Live B lymphocytes were assessed before and after maturation. Multivariate analysis was applied to distinguish cellular components within each cell. The spectral differences between non-activated and activated B lymphocytes were then identified, and their correlation with known intracellular biological changes were assessed in comparison to conventional RNA-seq analysis. Our data shows that spectral analysis provides a powerful tool to study gene activation that can complement conventional molecular biology techniques and opens the way for mapping the dynamics in the biochemical makeup of individual cells. The ability to measure and quantify molecular changes during cellular development can enable the characterization of cells during differentiation, cellular responses to extracellular cues, or disease progression. Conventional techniques, such as fluorescent tagging of molecules visualized with fluorescence microscopy  and tranVibrational spectroscopy exploits the interaction between light and molecules to probe their vibrational modes in order to obtain a “chemical fingerprint” of a sample. Both FTIR and Raman have been used to monitor modifications to or changes in expression of specific biomolecules, such as DNA levels during cell cycle , proteinIt is this label-free classification that has great potential both in (i) clinical settings, for disease diagnosis and prognosis, and (ii) in biomedical research, for example in cell sorting for downstream processes. How powerful this tool can be, depends upon our understanding of the correlation between the spectral output and the underlying biochemical pathways within the cells. In bacterial research, antibiotic resistance is of great interest. Spectral markers of antibiotic resistance have been identified at the population level , and morTranscriptomic readout consists of thousands of RNA transcripts, whereas Raman spectroscopy can measure the phenotypic expression of the RNA transcripts, i.e., the biochemical result of the transcriptomic profile. Noteworthy, all transcripts do not change independently; instead, strong correlations are found between transcripts that are controlled by global regulators, reflected in the Raman signals. In the yeast study, it has been determined that only 17 transcripts are sufficient for determining a linear correlation with the Raman spectra. The transcripts largely responsible for the linear correspondence have been identified by determining the variable importance in projection (VIP) values for each transcript. The top scoring transcripts were primarily non-coding RNAs in yeast and ribosome-related transcripts in bacteria . The corTo our knowledge (i) the correlation between Raman spectra and transcriptomic readouts has never been studied in mammalian cells, and (ii) it has not been examined in the context of cell differentiation. To explore this correlation in mammalian cells, B lymphocytes were chosen as a model cell system. Immune activation of these cells initiates large-scale changes to the transcriptomes, resulting in the differentiation of naïve B cells into mature B cells and class switch recombination (CSR) of the immunoglobulin receptor. Furthermore, as CSR requires reorganization of the DNA, it is a highly regulated process. A large number of regulatory proteins and RNAs have been shown to be involved . HoweverCH12F3 cells were cultured in RPMI 1640 medium with 10% fetal bovine serum, 5% NCTC-109, 1% Pen-Strep, 1% glutamine, 1% sodium pyruvate and 50 μM β-mercaptoethanol. The cells were immune activated by incubating them with a cytokine cocktail (CIT) consisting of 2.5 μg/ml anti-CD40, 10 ng/ml IL-4, and 50 ng/ml TGFβ.Flow cytometry measurements, for monitoring class switching assays of CH12F3 cells, were performed using a BD Accuri C6 Plus flow cytometer. 1% PFA fixed CH12F3 cells were stained with FITC Anti-mouse IgA antibody and APC Anti-mouse IgM antibody, both 1:200 dilution.A microfluidic silicon mold was designed to create a small reservoir chip for maintaining cell viability during Raman measurements as previously described . This mo–1), and Raman maps were acquired with a 200 nm step size (5 measurement points per micrometer) using a 0.1 s integration time per point. Cells were kept in the microfluidic chip for a maximum of four hours during measurements, before a new chip with fresh cells was prepared.Cells were washed in PBS, pelleted, and resuspended in PBS. PBS was flowed into the bonded chip using Portex tubing PE 0.86 × 0.33mm BxW using a syringe and a 21G microlance. Cells were flowed into the chip in the same way and left to settle for minimum 30 min. Raman maps were collected using a WITec alpha300R confocal Raman microscope system consisting of a 532 nm laser at 16 mW, a fiber-coupled UHTS spectrometer and an optical microscope with a 0.7 NA, 50 × objective. The microfluidic chip containing the cells in PBS suspension was held coverslip side facing up in a custom holder. Single cells (adhering to the glass) were identified in white light imaging mode, then the focus was adjusted in Raman mode using the oscilloscope to maximize the scattered signal intensity . This involved the calculation of similarity measures between each of the spectra from all 118 maps , each map was either 15 × 15 μm (75 × 75 pixels) or 20 × 20 μm (100 × 100 pixels). As the most similar spectra are grouped together, the spectrum of the new group becomes the mean of its members. At the end of the process, when ten similar group clusters remained, they contained the spectra from regions of cells with similar biochemical constituents, each represented by a mean spectrum or centroid. Each of the 10 clusters was assigned as nucleus, cytoplasm or background, based on the spectral profiles of the cluster centroids. All the spectra that made up the nuclear, cytoplasmic and background spectra were separately averaged for each map. Hence, a mean nucleus, cytoplasm, whole cell (cytoplasm + nucleus) and background spectrum was extracted from each cell map. An array of 13 maps  was discarded from the dataset before further analysis, since they either contained no pixels identified as nucleus or cytoplasm, or very few pixels associated with nucleus—with a nucleus size of less than 3 μm.–1). (2) The background spectra were smoothed using a Savitzky-Golay filter  to reduce the effect of noise. (3) The smoothed background spectra were then subtracted from the nucleus, cytoplasm and whole cell spectra.To remove the background signal (from the coverslip and PBS), the background spectra were subtracted from the nucleus and cytoplasm spectra for each map in three steps. (1) The spectra were baseline corrected by subtracting an offset  was also performed to calculate a supervised classification model based on a combination of the PC scores. The resulting linear discriminant function could be used to single out the key peaks responsible for the discrimination between D0 and D4 cells.Principal Component Analysis (PCA) was performed on nucleus, cytoplasm and whole cell spectra. For each principal component, a Total cell RNA was extracted by TRIzol followed with chloroform for phase separation and 100% isopropanol for RNA precipitation. Total RNA was eluted in 30 μl RNase-free water after being washed twice in 75% ethanol. The RNA concentration was assessed using a NanoDrop 2000 spectrophotometer . The RNA yield and size distribution were analyzed using an Agilent 2200 Tapestation with RNA Screentape .For small RNA library preparation, RNA aliquots were used for library preparation using NEBNext Multiplex Small RNA library preparation kit . The PCR amplified cDNA construct (from 140 to 160 bp) was purified using a QIAquick PCR Purification kit (Qiagen). The purified cDNA was directly sequenced using an Illumina MiSeq 2000 platform .® UltraTM RNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer’s instructions. Libraries were tested for quality and quantified using qPCR . The resulting libraries were sequenced on a HiSeq 2500 instrument that generated paired-end reads of 100 nucleotides.For long RNA library preparation, libraries were constructed using Ribo-Zero Magnetic Gold Kit (Human)  and NEBNextRaw sequencing reads were checked for potential sequencing issues and contaminants using FastQC. Adapter sequences, primers, number of fuzzy bases (Ns), and reads with quality scores below 30 were trimmed. Reads with a length of less than 20 bp after trimming were discarded. Clean reads were aligned to the mouse reference genome  using the TopHat 2.0 program, and the resulting alignment files were reconstructed with Cufflinks . The traP-value ≤ 0.05 and false discovery rate (FDR) ≤ 0.05 were considered statistically significant. Expression of significant differentially expressed genes in different B cell subsets was determined using My Geneset ImmGen  regression was applied to the datasets. The transcriptomic data consisted of the read counts for 17,725 transcripts with three D0 samples  and three D4 samples . Dimension reduced Raman data were used in the form of PC scores. To correspond to the three replicates for each condition of the transcriptomic data, the Raman cell measurements were randomly assigned to three groups of D0 and three groups of D4.i–) and transcriptomic (Ti–) datasets—meaning the PLS regression coefficients matrix, βi− was found, so thatPLS regression analysis was performed with a leave-one-out approach; each of the six samples was removed in turn. Each leave-one-out analysis determined a linear regression model between the Raman (Ri) and the β–i matrix.For each PLS regression analysis, Raman PC scores were predicted for the left-out sample, i, using the transcriptomic data , they were compared against the single cell PCA scores. For further assessment, the predicted Raman PC scores were then converted to LDA scores and again compared with the single cell data.Raman maps were collected from 118 live CH12F3 cells suspended in isotonic PBS-filled microfluidic chambers. The cells remained in place throughout measurements. Common k-means analysis was applied to identify cell (vs. background) pixels, as well as to distinguish the nucleus from the cytoplasm within each cell . AdditioThe quality of the segmentation was assessed in two ways. Firstly, the mean nucleus spectrum was compared to the mean cytoplasm spectrum . The mosTo assess large-scale chromatin conformational and transcriptomic alterations, two groups of CH12F3 cells were compared: non-activated cells (D0) and cells at 96 h post immune activation with cytokine  cocktail (D4), as shown in –1 shows a large variation between the two activation states. The neighboring peak at 752 cm–1 does not show this variation. Both of these peaks are associated with nucleic acids  and 752 cm–1  were amongst these, supporting the use of that peak ratio to distinguish between D0 and D4. Although a number of other nucleic acid peaks were identified, it is clear that intracellular changes of protein and lipid are also drivers for the spectral differences.To further explore the spectral differences between D0 and D4 cells, multivariate approaches were applied. An unsupervised method, Principal Component Analysis (PCA), showed a separation between D0 and D4 cells . Four PC–1 range are again present. The largest peaks include nucleic acid, protein, sugar and lipid, such as guanine and cytosine  was then applied, building on the PC scores and determining a classifier to discriminate between D0 and D4 cells. The two groups showed a very good separation . Using aIghm and Igha, which code for the immunoglobulin heavy chain constant regions of IgM and IgA, respectively, are highlighted. Ighm expression is down-regulated in D4, while Igha is up-regulated. This is a hallmark of the CH12F3 class switching response and in agreement with the IgM to IgA isotype switching measured by flow cytometry . Statistical significance was set at false discovery rate (FDR) < 0.05. As a result, we could observe that the three D0 samples cluster in proximity together, as do the three D4 samples. But the D0 vs. D4 clustered markedly separately from each other when plotted on the same graph  regression analysis was applied to create a model for the prediction of Raman data from transcriptomic data of CH12F3 cells. A PLS regression model was determined from three D0 samples  and three D4 samples  of transcriptomic and Raman data . Using aTo assess the validity of these predictions and thus the PLS regression model, the predicted Raman data were compared to the PC scores for each individual cell from the original PCA of the training cell dataset for each sample. Plotting the predicted PC scores against the single cell scores shows D0-2 and D4-2 within their expected regions  and the These results show that a linear correlation exists between transcriptomic profiles and Raman spectra of CH12F3 cells. Specifically, the variation in transcript expression levels between D0 and D4 cells is reflected in variation in Raman spectra of D0 and D4 cells—and transcriptomic data can be used to predict Raman data of CH12F3 cells.in vivo activated B cells isolated from murine germinal center splenocytes . We also identified the transcripts with the highest importance for the correlation with Raman spectra of non-activated and activated CH12F3 cells . The immst cells , our worMoving forward, it would be worth correlating Raman data to transcriptomic data in a reverse order to increase confidence of the data validity, but also to potentially provide a rapid, non-destructive, surrogate measure of potential transcriptomic profile from a known cell type. It would also be very interesting in the future to combine single-cell RNA-seq with Raman measurements to provide a more specific and accurate correlation. Especially, allowing us the potential to predict transcriptomic profiles from Raman imaging data, which would be an extremely powerful future Raman application tool. To accomplish this though, it might be important first to: (i) compare sufficient numbers of single-cell Raman datasets to a commensurate size of single-cell RNA-seq profiles, and (ii) isolate the nuclear vs. cytoplasmic signals to purity, through biochemical isolation of individual cellular nuclei via hypertonic solution cytoplasmic lysis. Ultimately, from the identification of differential Raman spectra differences and signatures, it could be possible to deduce correlating changes in gene expression, such as overall RNA sizes distribution and subtypes , and ultimately to predict expression of specific genes. This would be a very powerful approach to unraveling the direct relationship between the two complementary data types. Indeed, there is still much to be determined, but there most certainly is a correlation between transcriptomic profiles and Raman spectra and this correlation can be further enhanced by combining more refined biological techniques. Understanding the origin of this correlation in different case studies will add value to Raman measurements of biological samples and aid interpretation of spectral changes. Furthermore, the work outlined here, suggests that Raman may also aid in the identification of key regulatory transcripts for immune activation. Future work will demonstrate if this can be translated to elucidating other cellular developmental processes, occurring in healthy cells or during disease.https://doi.org/10.6084/m9.figshare.14135219.v1.The data presented in the study are deposited in FigShare, FP, RC, SP, and NS conceived and designed the experiments. RM and SP constructed the microfluidic chip. RM performed the experiments and wrote the manuscript with inputs from the other authors. KY acquired the transcriptomic data. RM, KY, and NS analyzed the data. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


KRWs with RDs showed a significantly lower incidence of weight loss than those without RDs . Class 1 KRWs and exclusively staffed RDs were independently associated with lower odds of weight loss . Approximately 18% of KRW patients lost weight, and having RDs on staff can lower the risk of weight loss.There is scarce evidence regarding the risk of weight loss and the effect of having registered dietitians (RDs) on staff in rehabilitation wards on weight loss. We aimed to examine the effects of RDs in Kaifukuki  rehabilitation wards (KRWs) on the prevention of weight loss in adult patients. Data from 2-year nationwide annual surveys on KRWs in Japan were retrospectively analysed. Weight loss was defined as loss of ≥5% weight during the KRW stay. Risk of weight loss in class 1 KRWs (obligated to provide nutrition care) was compared with that in class 2–6 KRWs (not obligated). Risk of weight loss in class 2–6 KRWs with RDs was compared to those without. Overall, 17.7% of 39,417 patients lost weight. Class 1 KRWs showed a lower risk of weight loss than class 2–6 KRWs (17.3% vs. 18.5%,  Malnutrition is often seen in older adults and can result in reduced functional capacity. Up to 10.5% of community-dwelling seniors, 7.7% of nursing home residents and 14% of hospitalised patients experience unintentional weight loss ,3,4,5. MTo prevent malnutrition and regain nutritional status, inpatient rehabilitation should involve nutritional support as part of a multidisciplinary approach. For example, inpatient rehabilitation facilities in the US are obligated to provide a nutritional assessment within 3 days of admission . AnotherLoss of body weight is one of the key features of malnutrition; it hampers functional recovery and may be partially preventable with nutritional support ,11,15. SThus, this study aimed to explore the risk of weight loss in KRWs and examine the effects of having RDs in KRWs on the prevention of weight loss in adult patients in the real world using a nationwide survey. The KRW Association is the sole association of which 84% of the hospitals with KRWs are members , and it We obtained the patient-related information that was linked to corresponding hospital- and ward-related information from the dataset of the two-year survey results. All the data from patients aged ≥20 years were included. Data with missing values of height, body weight, FIM, number of RDs on staff and the KRW class were excluded. Data from patients who stayed in the KRWs ≤30 days , patientStaff RDs were defined by the number of RDs who worked at the ward. They were assigned to one of two categories: (1) the number of RDs who worked at the ward but also completed other tasks such as food service or nutrition management in other wards simultaneously (staffed RD) and (2) the number of RDs who worked at the ward but did not complete other tasks (exclusively staffed RD). In the survey form, ‘staffed RD’ was defined as spending at least 50% of working time in providing nutrition care in the assigned ward, whereas ‘exclusively staffed RD’ was defined as spending ≥80% of working time in providing nutrition care in the ward.The RDs must adhere to the following regulations when providing nutrition care in KRWs: (1) Obligations; nutritional assessment; participating in preparing a rehabilitation plan; nutritional care planning; nutritional monitoring; nutritional education. (2) Recommendations; nutritional screening; collecting the data on activities of daily living and swallowing function; calculating nutrient requirements; participating in rehabilitation conference; arranging nutritional care plan as a part of discharge planning. Details of the obligations and recommendations for RDs in the KRWs are shown in 2; normal weight, 18.5 ≤ BMI < 23 kg/m2; overweight, 23 ≤ BMI < 27.5 kg/m2; obese, BMI ≥ 27.5 kg/m2. The primary disease/conditions were classified into four categories based on the national healthcare insurance policy: stroke; other neurological diseases/injuries; orthopaedic disease/injuries; and hospital-associated deconditioning. Body mass index /height (m) squared) was categorised into four groups in accordance with the WHO recommendation for the Asian population : underwe2; this information was provided as part of the patient-related information in the survey form.The primary outcome of this study was the loss of body weight during the KRW stay, which was defined by at least a 5% decrease in body weight at discharge compared with body weight measured on admission to the KRW. The percentage of weight loss was calculated by subtracting the body weight at discharge from that on admission. Since the upper limit of the length of stay was 180 days (6 months), 5% or more weight loss can be considered as a phenotype of malnutrition . Focusint-test, while those with non-normal distribution were performed using a Mann–Whitney U-test. Categorical variables were tested statistically using a chi-squared test or Fisher’s exact test. Significance level was set at p < 0.05. Multiple comparisons performed using Bonferroni correction were performed if there was a statistically significant difference for multi-group categorical variables.All statistical analyses were performed by JMP 11.2.1 software . Normally distributed variables were presented as means and standard deviations, whereas skewed variables were expressed as medians and interquartile ranges (IQR). Qualitative variables were reported as numbers (%). The hypothetical test for quantitative variables with normal distribution was performed using a Outcome measurements were compared through two separate analyses: (1) Comparison between class 1 KRWs (obligated to nutrition care) and class 2–6 KRWs (not obligated to nutrition care); (2) Comparison between class 2–6 KRWs with and without exclusively staffed RDs. All class 1 KRWs provided the aforementioned nutrition care for relevant patients, and most of them had RDs on staff; therefore, the effectiveness of nutrition care could be tested in Analysis 1. In Analysis 2, the real effect of ‘exclusively staffed RDs’ could be examined because the background of the wards regarding nutrition care other than RDs was similar in classes 2–6. A binary logistic regression analysis was performed to test the association between the incidence of weight loss and the KRW class or having RDs on staff. To adjust for the confounding effect of BMI at discharge, multiple linear regression analysis was performed. Potential confounders for these analyses were selected from the previous literature and biological plausibility : age  and the requirement for informed consent was waived because the data on the survey response were all anonymised.Among them, 25,324 and 14,093 were admitted to class 1 KRWs and class 2–6 KRWs, respectively. The class 1 KRW group included a greater proportion of stroke patients and lower proportion of orthopaedic patients than the class 2–6 group. The former group also showed a shorter length of KRW stay, a lower FIM and a lower proportion of underweight patients than the latter group. Regarding nutrition care, 61.0% of the patients in the class 1 KRW group received nutrition monitoring at least once a week and 24.8% received nutrition counselling during hospitalisation.p = 0.003).Of the 39,417 underweight or normal weight patients, 17.7% of them experienced weight loss overall. The proportion of patients who experienced weight loss was significantly lower in the class 1 KRW group than in the class 2–6 KRW group .p = 0.015).p = 0.006). Additionally, age, male sex, diseases other than stroke, days between onset and admission, initial FIM, initial BMI and daily rehabilitation dose were also significantly related to weight loss.p = 0.011) as well as male sex, diseases other than stroke, longer duration (days) between onset and admission, higher FIM on admission and greater dose of daily rehabilitation. Conversely, older age and higher body weight were associated with higher odds of weight loss. p = 0.129) was not independently associated with BMI at discharge. There was a positive relationship between BMI at discharge and other neurological diseases/injuries, days between onset and admission, FIM at admission, BMI at admission and daily rehabilitation dose. In contrast, older age was inversely related to BMI at discharge. There were two major findings from this retrospective analysis of a nationwide survey. Firstly, 17.7% of the normal or underweight patients experienced a loss of body weight during their KRW stay. Secondly, the obligation to provide nutrition care and exclusively staffed RDs in the KRW were positively associated with a lower incidence of weight loss.2, suggesting that these studies included obese patients who required weight loss as a part of their treatment. On the other hand, a longitudinal study of stroke patients also showed no weight loss during 104 days of rehabilitation after approximately 14 days from the onset of stroke  [To date, several studies have reported changes in body weight during rehabilitation, but no studies have focused on patients without obesity ,16,26,278 kg/m2) . Poor ph8 kg/m2) . Indeed,8 kg/m2) , a suffiIn our analyses, more than 80% of the class 1 KRWs that were obligated to provide nutrition care had RDs on staff, and they showed a significantly lower incidence of weight loss than the class 2–6 KRWs. More importantly, having exclusively staffed RDs in the class 2–6 KRWs was negatively associated with weight loss. These findings are consistent with previous studies ,31 and sOne of the strengths of our study was its analysis of a large number of samples in the real-world setting from the national survey. To the best of our knowledge, this is the first study to examine weight loss during inpatient rehabilitation using nationwide samples. As described, the original datasets covered 62–65% of all KRWs in Japan and there seems to be a small amount of bias regarding regional variety due to the location of the KRW. Additionally, Japan applied universal healthcare insurance coverage for all people living in Japan , and conHowever, there were some limitations to this study. First, the pathophysiology of malnutrition, as well as the actual nutritional status of patients and details of the nutrition care provided, could not be analysed because the original datasets did not contain this information. Because the current consensus on the definition of malnutrition relies on both phenotype  and aetiology  , actual This retrospective analysis of a nationwide survey of KRWs in Japan revealed that 17.7% of underweight or normal weight patients lost more than 5% of their body weight during hospitalisation. In addition, the KRWs obligated to provide nutrition care and with exclusively staffed RDs were able to significantly reduce the incidence of weight loss. Further research is needed to clarify the actual changes in nutritional status during the KRW stay, and the effect of RD practice on improvements in nutritional disorders.


Radiosynoviorthesis (RSO) using the intraarticular application of beta-particle emitting radiocolloids has for decades been used for the local treatment of inflammatory joint diseases. The injected radiopharmaceuticals are phagocytized by the superficial macrophages of the synovial membrane, resulting in sclerosis and fibrosis of the formerly inflamed tissue, finally leading to reduced joint effusion and alleviation of joint pain.The European Association of Nuclear Medicine (EANM) has written and approved these guidelines in tight collaboration with an international team of clinical experts, including rheumatologists. Besides clinical and procedural aspects, different national legislative issues, dosimetric considerations, possible complications, and side effects are addressed.These guidelines will assist nuclear medicine physicians in performing radiosynoviorthesis. Since there are differences regarding the radiopharmaceuticals approved for RSO and the official indications between several European countries, this guideline can only give a framework that must be adopted individually. Intraarticular therapy using colloidal beta-emitting radionuclides, radiosynoviorthesis or RSO, is known for almost 70 years and is indicated in patients suffering from various inflammatory joint diseases . The pre90Y]yttrium citrate, [186Re]rhenium sulfide, and [169Er]erbium citrate deliver their high-energy beta particles to the innermost cell layer of the synovium, leading to pronounced cell death, obliteration of capillary blood supply, and hence fibrosis and sclerosis of the synovial membrane  is below 0.1 per 1000 . The radThis text updates and replaces the 2003 EANM guideline on radiosynoviorthesis and contains general information regarding the different indications for radiosynoviorthesis and contraindications, necessary pre-therapeutic diagnostics, and the treatment procedure itself. Moreover, essential aspects of this therapy, like dosimetry, radiation burden, legislative issues, and possible complications and side effects are discussed . FinallyHowever, this document does not claim universal validity, especially legislative issues regarding indications, approval of the different radionuclides, or radiation protection rules may vary between different countries and must be followed according to the national laws.Radiosynoviorthesis is an important technique for treating mono- or oligoarticular synovitis, and many factors may influence its effectiveness and safety. This project aims to establish recommendations to standardize indications and procedures to improve effectiveness and safety.A best-evidence review of PubMed articles published until 01/09/2020 was performed to obtain efficacy outcomes of RSO in various indications. Selection criteria included English or German language studies considered to be most relevant  to the clinical application(s) in question. It is recognized that limiting the search criteria to English and German language publications may introduce bias, but this can be justified because most RSO treatments are performed in Germany. Case reports and case series with a sample size less than 10 (arbitrarily chosen) were excluded.90Y]yttrium citrate for the treatment of knee joints only, [186Re]rhenium sulfide for mid-sized joints, and [169Er]erbium citrate for small joints. The current official approvals and marketing authorizations in European countries are given in Table Radiosynoviorthesis using an intraarticular injection of beta-emitting radiocolloids is a local treatment in patients suffering from inflammatory joint disease. Depending on the joint’s size, three radiocolloids with different physical properties are used: yttrium citrate is indicated in adults for the therapeutic irradiation of synovial hypertrophy of the knee joint mainly for mono- or oligoarthritis of chronic inflammatory rheumatic disorders, in particular rheumatoid arthritis and in hemophilic arthropathy.[186Re]rhenium sulfide is indicated for the treatment of rheumatoid mono- or oligoarthritis involving medium-sized joints, including rheumatoid arthritis, hemophilic arthropathy, and chronic arthropathy associated with articular chondrocalcinosis.[169Er]erbium citrate is indicated for the treatment of rheumatoid mono- or oligoarthritis of small joints of hands and feet following the failure of intraarticular corticosteroid therapy or when the latter is contraindicated.erbium citrate is approved for the first two indications only. In Switzerland, [186Re]rhenium sulfide is also approved to treat the knee joint.erbium citrate, [186Re]rhenium sulfide, and [90Y]yttrium citrate yttrium colloid was superior to placebo administration (odds ratio (OR) 2.42, 95% confidence interval (CI) 1.02–5.73) for achieving treatment success, but possible publication bias could not be ruled out. [90Y]yttrium colloid was not superior to triamcinolone  [90Y]yttrium colloid synovectomy deserves a place in clinical practice” [186Re]rhenium colloid and that it is a suitable second-line treatment for RA patients in whom other therapies (including GC injections) have failed, with success rates varying between 34 and 94% [186Re]rhenium colloid and [169Er]erbium colloid combined were 2 (95% CI 0.66–6) and 2 (95% CI 1.09–3.5) at 6 and 12 months, respectively [Several reviews with and without meta-analyses have been published –39. Jone1–10.55) . Deutsch1–10.55) . The overactice” . However and 94% . Van der90Y]yttrium colloid + GC and [153Sm]samarium colloid + GC to GC alone (control group). Only for the pain parameter, [90Y]yttrium colloid + GC proved superior to [153Sm]samarium colloid + GC and GC at 1 week, and [90Y]yttrium colloid + GC proved to be superior to GC at 48 weeks. Furthermore, the study concluded that there is no indication for RSO of the knee with [153Sm]samarium colloid in RA patients [90Y]yttrium colloid as well. Of the studied patients, 11 had RA. At 6 months, there was an improvement in swelling, tenderness, and pain. No short-term side effects or complications were recorded [90Y]yttrium colloid remains a safe and effective treatment in an era of improved DMARDs. The clinical response in 46 RA knee joints was mild, moderate, and complete in 24%, 26%, and 28% at 3 months, respectively. However, at 36 months, the response declined, especially in mild responders [90Y]yttrium hydroxyapatite found a reduction of pain, swelling, and improvement of knee motion in approximately 80% at 6 months and 76% at 12 months [90Y]yttrium colloid, including 73 RA cases, pain relief was observed in 81% at 6 months (33% with complete pain relief) and in 87% at 1 year. The likelihood of pain relapse in RA cases was 15% at 6 months and 27% at 12 months [90Y]yttrium colloid, 15 with [32P]phosphorus colloid, and 16 with [188Re]rhenium colloid) were compared with 46 intraarticular GC injections. Pain relief at 3 months was 86% for the RSO group versus 67% for the GC group. At 6 and 12 months, the numbers were 72% versus 46% and 46% versus 21%, respectively. There was no significant difference between the three radiocolloids [32P]phosphorus colloid in RA patients reported excellent response in 56%, moderate response in 9%, and poor response in 35% [177Lu]lutetium hydroxyapatite [169Er]erbium, [186Re]rhenium, and [90Y]yttrium colloid, in 577 joints of 137 RA patients, Liepe et al. reported success rates (excellent or good response) in 57% of the treated knees, 63% of shoulders, 60% of wrists, 64% of ankles, 54% of thumb bases, 55% of MCPs, 54% of PIPs, 53% of DIPs, and 54% of MTPs phosphorus colloid rhenium colloid was used, but similar results can be expected with the approved [186Re]rhenium colloid.Therefore, RSO should be used as early as possible before developing hemarthropathy , 58. TheIn pediatric hemophilia, RSO is indicated when chronic persistent or recurrent synovitis with or without recurrent joint bleeding (2–3 episodes per 6 months) is detectable. It can also be used when moderate hemarthropathy exists and surgical interventions are not yet required. RSO is an option when inhibitors against factor VIII/IX are present , 78 or wSimilar to rheumatoid arthritis, RSO could be helpful in selected patients with osteoarthritis and proven synovitis, resistant to other therapies; however, due to national rules, it has to be done as an off-label use in some countries.Osteoarthritis (OA), also known as degenerative joint disease, is a large and increasing burden for the health systems in many countries. In Europe, more than 30 million people might be affected. Similar data with an increased prevalence of osteoarthritis is also seen worldwide in an aging population . BesidesMost often, the weight-bearing joints of the lower extremities are affected. In finger OA, the distal interphalangeal (DIP) joints, proximal interphalangeal joints (PIP), and the first carpometacarpal (CMC) joints are frequently involved.Symptoms include joint pain, stiffness, and sometimes a joint effusion. Joint pain is the leading clinical symptom of OA. It was discussed in the literature that the intensity of the pain depends on the radiological degree of bone alterations in the affected joint. Additionally, recent animal studies demonstrated that blood vessels with nociceptive nerve fibers penetrating the osteochondral junction into the cartilage might contribute to the joint pain . Furthern = 72), an excellent/good response was observed in 45.9% and 41.2% after 8 years.In the literature, the improvement rate after RSO ranges from 40 to 89%. In a recent long-term prospective study of RSO in synovitis in OA of the knee joint, Szentesi et al.  reportedIn a meta-analysis of 121 patients with OA treated with RSO, the mean success rate was 56 ± 11% erbium colloid combined with GC was compared to GC injection alone with 1 year of follow-up. RSO combined with GC resulted in significant reductions of pain, inflammation, and improvement of motion.Another group performed a double-blind controlled prospective study on 22 patients with local synovitis in OA of the thumb base joints. The effect of yttrium citrate. In 93/107 (87%) of patients, the joint effusion resolved entirely after therapy.RSO was also studied for treating recurrent joint effusions after knee arthroplasty. However, only scarce data are available in the literature. The initial results with RSO in knee arthroplasty were reported by Mödder et al. . In thei90Y]yttrium colloid technetium nanocolloid) injected into the knee joint is detected in the popliteal soft tissue surrounding Baker’s cyst after movement of the joint, a rupture is assumed, and the RSO is strictly contraindicated in this patient. However, since radiocolloids are not officially approved for intraarticular application, the patient must provide informed consent that this examination is an off-label use.This technique can also be used to exclude a rupture of Baker’s cyst of the knee in selected patients. If leakage of a radiolabelled colloid  strongly correlates with pannus volume as evaluated at MRI and shows a correlation with clinical symptoms of arthritis such as tenderness and swelling yttrium colloid into breast milk was shown [186Re]rhenium colloid and [169Er]erbium colloid; thus, the same procedure is advisable and is given in the SPC.Breastfeeding should be terminated prior to RSO. A transfer of yttrium colloid erbium colloid, the same effect of EDTA-containing contrast media was described and thus, the amount of intraarticularly injected contrast agents during RSO should be as low as possible rhenium sulfide with a microcrystalline sustained-release glucocorticosteroid should be avoided in the hip due to the possibility of femoral head necrosis after local injection, regardless of the preparation used.Subsequent injection of a glucocorticosteroid: preferably triamcinolone hexacetonide, triamcinolone acetonide, or betamethasone. The combination of yttrium colloid could lead to severe complications such as skin atrophy or even soft tissue necrosis.The needle’s intraarticular positioning is checked either by the presence of a synovial effusion, even if minimal, before any injection, or by a small intraarticular injection of contrast agent in the absence of effusion. A paraarticular injection of erbium colloid. This is relatively easy by exerting traction in the axis of the finger one can, most often, open the joint and locate the puncture point.The injection in the metacarpophalangeal joint is done with a shorter needle of 25 Gauge and 16 mm. The same needle will be used for the small joints of the fingers to inject rhenium colloid can be injected without pressure and pain.Hip RSO can be done under fluoroscopy or ultrasonography. Because of simplicity, the anterior approach under fluoroscopy is preferred. The patient is lying in a supine position; the feet are in internal rotation. A marker could be placed on the skin on the external two-thirds of an imaginary line that goes from the pubis to the greater trochanter. The presence of the iliac neurovascular bundle is checked by palpation, well within the puncture site. A 21 Gauge 50-mm or 20 Gauge 9–10-mm needle is used, positioned at 90° to the plane of the thigh. After bone contact, the needle is withdrawn by a few millimeters. Fluoroscopy is used to confirm the correct positioning of the needle in the middle of the femoral neck and [186Re]rhenium sulfide, followed by a corticosteroid, is injected without pressure.For the shoulder joint, namely the glenohumeral cavity, three approaches are preferred: the anterior, the upper, and the posterior approaches. It is preferred to position the patient in a supine position, arm in external rotation, and inject through the anterior route. A marker may be placed on the skin after palpation of the joint space by mobilizing the arm. The needle must be positioned in the lower third of the joint space. A 50-mm or 40-mm, 21 Gauge needle is most often used. The needle penetrates the skin at 90° to the plane of the shoulder. Local anesthesia is sometimes useful because the procedure can be painful, especially in contact with the periosteum. Once in contact with the joint capsule, a small internal rotation of the humeral head may be required to allow the needle to slide into the glenohumeral joint. Sometimes an effusion is found and evacuated. If there is no effusion, the correct positioning of the needle is checked by fluoroscopy. The radiopharmaceutical yttrium citrate is used for knee joints and [186Re]rhenium sulfide for medium-sized joints. Country-specific requirements or approvals must be observed . The selection of radionuclide and the applied activity depend on patient weight and age, joint size, and the thickness of the synovium in children. For knee joints, the procedure could be performed without fluoroscopy solely, while in all other joints, fluoroscopy, and contrast medium arthrography are advisable, especially in case of a narrow joint space or more extensive osseous deformities rhenium colloid is to be used for RSO of a knee joint in smaller children should be decided based on the individual case. Despite the reduced depth of penetration compared to yttrium-90, it can be assumed that the same effect can be achieved with less radiation exposition of the joint and the surrounding tissue. However, due to the good long-term experiences in hemophiliac patients, [90Y]yttrium colloid could also be considered for treating the knee in the pediatric population yttrium colloid, 444 MBq for [186Re]rhenium colloid, and 300 MBq for [169Er]erbium colloid per year.RSO can be repeated after 6 months, if a sufficient therapy success has not been achieved after the first application. A visual analog scale (VAS), for example, is useful for evaluating the success of treatment based on subjective pain sensation. If the pain symptoms are not at least 50% lower, the therapy success is not sufficient. The shortest interval for repeating RSO is at least 6 months according to the technical information and instructions for use. However, the approved maximum annual cumulative administered activity of the respective agent must be observed. For example, in Germany, the maximum cumulative activity is limited to 444 MBq for rhenium colloid (gamma component) and [90Y]yttrium colloid (Bremsstrahlung), the intraarticular radionuclide distribution should be documented immediately by scintigraphy, in case of [90Y]yttrium colloid also by PET/CT. Moreover, any extra-articular radionuclide distribution in case of a paraarticular  injection can be excluded by this procedure. Imaging 48 h after RSO could be helpful to detect lymphatic drainage from the treated joint , representing the equivalent dose at a depth of 0.07 mm in the body at the position of the partial body dosimeter, is particularly relevant for radiation protection. As known, beta radiation can be shielded well by materials with a low atomic number, for example, by syringe shields made of acrylic glass . As an approximation, the thickness Concerning radiation protection, the special properties of beta emitters must be taken into account during the application, which due to their short range can cause a high surface dose. The surface personal dose HIn addition to the consistent use of such shields, distance-increasing devices must be used to prepare the syringes and apply the radiopharmaceutical. For this purpose, it is recommended to use additional holding forceps or tweezers to fix the cannula  or to usAll syringes prepared for one RSO setting must be stored in a plastic box with adequate shielding before use. The waste should be collected with clear-cut separation between the three radionuclides used. Their different half-lives determine the clearance of the waste for its final disposal according to national regulations.186Re]rhenium and 9 Gy with [169Er]erbium [90Y]yttrium-based RSO to reach similar effects as surgical synovectomy [The options for individual determination of the absorbed dose to the treated synovium and cartilage are limited. A 2-D Monte Carlo model for the synovial joint has been developed for calculating depth-dose profiles for source activity distributed in the synovial fluid . The absr]erbium . This vaovectomy . The dosovectomy , 134. An90Y]yttrium Bremsstrahlung SPECT/CT and PET/CT has been demonstrated for verifying uniform distribution and target dosimetry rhenium sulfide is impossible due to high variability in injected joints, activities, and immobilization strategies. The available data mainly measured activity leakage from RSO of the ankle and the upper extremity joints. The total mean leakage ranged from 2.5 to 3.9% yttrium is the most potent radionuclide to induce tissue damage, possibly even full-thickness skin necrosis [186Re]rhenium colloid are much more limited, usually self-healing within 3–4 weeks, but in severe cases, hyperbaric oxygen treatment is helpful [169Er]erbium colloid, with its low energy and small tissue penetration, is unlikely to cause more profound necrosis, and beta radiation burns associated with its use require only conservative treatment yttrium, 4% with [186Re]rhenium, and negligible for [169Er]erbium colloid [90Y]yttrium, which is not considered sufficient to cause significant bone damage or necrosis. On the other hand, recent studies suggest that in patients with end-stage osteoarthritis and extensive cartilage erosion, beta radiation may cause avascular necrosis of the exposed subchondral bone phosphorus or [90Y]yttrium colloid revealed no increase in cancer risk compared to the general population [90Y]yttrium colloid.There have been two cases of acute lymphocytic leukemia reported in hemophilia patients receiving phosphorus-32. However, due to the short interval between radiation exposure and malignancy, the causal relationship between phosphorus-32 and leukemia development cannot be established in these patients. Moreover, both children had other autoimmune disorders , 183. Whpulation . Similarpulation . That st90Y]yttrium or [186Re]rhenium colloid [An increased risk of cancer after RSO with radiocolloids has not been reported. Many studies reported cytogenetic analyses, such as chromosomal aberration analysis, micronuclei, and sister chromatid exchange, to indicate radiation-induced cytogenetic damage in hemophilic children undergoing RSO with [ colloid , 185–188There is a risk of bleeding in patients with hemophilia due to puncture, which is very small if there is sufficient factor substitution . It is eThe key feature of arthritic conditions is primarily the presence of synovitis. Joint involvement and distribution depend on the type of arthritis. The treatment options for patients with arthritis have increased considerably within the last decades, especially with the introduction of biological disease-modifying anti-rheumatic drugs (bDMARDs) in the late 1990s. Recommended treatment strategies include early and aggressive treatment with mono- or combination therapy using first conventional synthetic DMARDS followed by bDMARDs. The clinical treatment goal is to obtain rapid disease control and suppress inflammation, thereby preventing pain and joint destruction and improving daily function and quality of life. This strategy was first outlined in the European League against Rheumatism (EULAR) 2010 treat-to-target recommendations where the treatment target was specified as clinical remission or at least low disease activity . DiseaseThe use of intraarticular glucocorticosteroid injection is a standard treatment in rheumatological practice combined with DMARDs in the early phase of the disease and as a treatment for joints with flare. In a randomized controlled trial of early RA patients, aggressive step-up treatment with methotrexate combined with intraarticular betamethasone (in a maximum of 4 joints per visit) produced rapid and effective disease control . RepeateDespite new drugs and aggressive treatment strategies, including the treat-to-target approach, some patients have persistent synovitis in a single or a few joints where surgical synovectomy or RSO may be indicated. RSO appears to perform better in RA patients than in OA patients , 94, 191While many studies have investigated the effect of RSO compared with intraarticular GC injection, their findings have been conflicting due to heterogeneity between trials. There are major differences in administering the radionuclide with or without GC, variability in the type of GC, and very important differences in the patient inclusion criteria. Furthermore, differences in clinical outcomes, including the lack of validated methods for measuring the clinical effect of RSO, make a comparison between studies difficult. Given that synovitis refractory to intraarticular GC treatment is a common clinical problem in rheumatology, RSO provides an additional treatment alternative to surgical synovectomy. Further studies are needed to establish wherein the treatment algorithm of RA RSO has its best place.For hemophilic patients, RSO has a clearly defined place in the treatment algorithm with persistent chronic synovitis after intensified factor therapy over 6 months respectively 3 joint bleedings per year, as supported by other guidelines .This guideline summarizes the views of the EANM bone and joint committee. It reflects recommendations for which the EANM cannot be held responsible. The recommendations should be taken into context of good practice of nuclear medicine and do not substitute for national and international legal or regulatory provisions.


Caffeine citrate is the drug of choice for the pharmacological treatment of apnea of prematurity. Factors such as maturity and genetic variation contribute to the interindividual variability in the clinical response to caffeine therapy in preterm infants, making the optimal dose administered controversial. Moreover, the necessity for therapeutic drug monitoring (TDM) of caffeine is still worth discussing due to the need to achieve the desired target concentrations as well as concerns about the safety of higher doses. Therefore, we reviewed the pharmacokinetic profile of caffeine in preterm infants, evidence of the safety and efficacy of different doses of caffeine, therapeutic concentration ranges of caffeine and impact of genetic variability on caffeine therapy. Whereas the safety and efficacy of standard-dose caffeine have been demonstrated, evidence for the safety of higher administered doses is insufficient. Thus, preterm infants who lack clinical response to standard-dose caffeine therapy are of interest for TDM when dose optimization is performed. Polymorphisms in pharmacodynamics-related genes, but not in pharmacokinetics-related genes, have a significant impact on the interindividual variability in clinical response to caffeine therapy. For preterm infants lacking clinical response, how to develop individualized medication regimens for caffeine remains to be explored. Apnea of prematurity (AOP), classified as central, obstructive, or mixed, is usually defined as a cessation of breathing in a premature infant for 20 s or longer, or a shorter pause accompanied by bradycardia (<100 bpm), cyanosis, or pallor . It is aSeveral interventions decrease apneic event frequency and duration. These include respiratory interventions including continuous positive airway pressure and pharmacologic therapies, such as methylxanthines, which have been used for over 40 years . CaffeinDespite caffeine’s frequent use in routine neonatal practice, there are controversies surrounding this medicine, which future researches may resolve, including the optimal dose of caffeine administration  and therMost of the pharmacokinetic (PK) studies for caffeine were performed in premature neonates . Due to The metabolism of caffeine occurs primarily in the liver. In adults, with the catalysis by CYP2A1 and CYP2E1, caffeine undergoes 1-, 3-, and 7-demethylation to generate the biologically active metabolites theophylline, theobromine, and paraxanthine, which can then be further demethylated to monomethylxanthine . DimethyThe serum half-life of caffeine in preterm infants is prolonged more than ten times that of adults because of immature hepatic metabolism and renal excretion . Caffeinn = 16 to n = 23), and the therapeutic effect of caffeine on AOP was observed by comparison with placebo or theophylline  for periextubation management of 127 preterm infants < 32 weeks gestation who were ventilated for > 48 h and found that there was no statistically significant difference in the incidence of extubation failure between different dosing groups . HoweverSeveral findings also revealed the benefits of higher doses of caffeine for VLBW preterm infants. A retrospective analysis suggested that a higher average daily dose of caffeine citrate was associated with better neurodevelopmental outcomes in VLBW infants . AnotherFour meta-analyses also synthesized the findings from the trials comparing higher and lower doses of caffeine citrate . Among op = 0.1) and burden   in infants < 31 weeks’ gestation who were randomized to a higher-dose caffeine citrate (loading 80 mg/kg) . Furtherp = 0.1) . These rp = 0.1) . The useCollectively, most previous RCTs had small sample sizes, and only two of them have reported 2 years clincal outcomes, which, although positive, need to be treated with caution. Thus, whether to use higher caffeine dosage regimens and how to optimize the caffeine dose are still questionable.The role of TDM for the control of therapeutic ranges of caffeine has often been challenged due to its benign safety profile when standard dosing is used. As early as in 1977, Aranda et al. revealed that the plasma concentration of standard dose caffeine needed to be monitored and the effective therapeutic concentration was established at 5–20 mg/L by referring to the use of theophylline . SubsequBlood caffeine levels in preterm infants were almost within this conventional target range in other studies using similar standard dose regimens. In an RCT, 37 preterm infants rapidly achieved the therapeutic concentration within 24 h after starting treatment with a significant reduction in apneic episodes . A studyn = 94) who lacked clinical response and had median to 75th quartile of plasma caffeine concentrations of 10.2–14.1 mg/L, suggesting that some neonates may need higher targets of caffeine to control apnea  was used, the serum drug concentrations were maintained in a safe therapeutic range and were independent of corrected gestational age, weight, and postnatal age within the first 2 weeks of life . Researches to date are precisely based on these two aspects.Earlier studies have found that heritability impacts the incidence of AOP, which raised interest in elucidating the effects of genetic factors on AOP as well as caffeine therapy . The the0/D ratio and genetic variations in CYP1A2 genes (rs2472299 and rs762551)  and PK parameters (AHR) CC genotype (rs4410790) differed significantly between the two groups with different responses to caffeine treatment in Chinese preterm neonates  . Corresprameters . These fneonates . Althoug1, A2A, A2B and A3, which is encoded by the ADORA1, ADORA2A, ADORA2B, and ADORA3 genes, respectively  CC genotype carriers had better responsiveness to caffeine than CT or TT genotype carriers.They also revealed that the correlation between ADORA2A  CT or TT genotypes and vulnerability to AOP as well as the correlation between ADORA2A (rs35320474) CT or TT genotypes and greater risk of BPD (ADORA2A (rs5751876) CT, TT genotypes and T allele in caffeine nonresponders compared to caffeine responders was also reported in another prospective case-control study of Egyptian preterm neonates (ADORA1 T > G (rs10920568), G > T (rs12744240) and ADORA3 C > A (rs10776727) as well as T > C (rs2298191) mutant genotypes did not respond to caffeine treatment, whereas ADORA2A T > A (rs34923252) and A > C (rs5996696) mutation genotype carriers responded better  responded poorly to standard-dose caffeine treatment  gene polymorphisms are the most described genetic factors in those current studies. AR is a class of G protein-coupled receptors with four known subtypes, Aectively . With a trations . In sometrations . Referrik of BPD . A signineonates . Moreoved better . In addid better . Phosphoreatment .Collectively, although the sample size number of these studies is small, several genetic polymorphisms have been revealed to be associated with individual variances in response to caffeine therapy. Therefore, studies with larger sample sizes are needed to confirm these findings and further researches are warranted to explain how genetic variants play a critical role in the response to caffeine therapy in premature infants.Caffeine is effective in reducing apnea frequency in preterm neonates. The available evidence has confirmed the efficacy and safety of standard doses of caffeine, and routine TDM seems unnecessary in neonates who respond positively to caffeine treatment. However, the well-known CAP trial only started caffeine treatment when apnea occurred, and when to start standard-dose caffeine therapy is also a quite controversial issue that requires long-term safety studies. For developmental premature infants, a dosing adjustment strategy based on postnatal age was proposed to maintain stable caffeine concentrations, and individualized caffeine medication may be administered with the help of PPK and PBPK models. For neonates lacking a positive clinical response, as the evidence for the use of higher doses of caffeine is insufficient, and TDM should be performed to achieve the desired blood caffeine level and ensure safety. The long-term results of larger trials of higher doses of caffeine are expected and would be more reasonable if corresponding blood caffeine concentrations could be provided. In addition, the study of genetic factors has preliminarily revealed the association between genetic polymorphisms and clinical response to caffeine therapy. Further studies are required to explain how genetic variants play a role in the response to caffeine therapy in premature infants. And how to establish an approach to individualize medication regimens for infants with poor clinical response by integrating tools such as TDM, genetic testing, PPK and PBPK models is also a direction for future exploration.


This study aimed to evaluate the incidence of chronic groin pain (primary outcome) and alterations of sensitivity (secondary outcome) after Lichtenstein inguinal hernia repair, comparing neurectomy with ilioinguinal nerve preservation surgery.The exact cause of chronic groin postoperative pain after mesh inguinal hernia repair is usually unclear. Section of the ilioinguinal nerve (neurectomy) may reduce postoperative chronic pain.We followed PRISMA guidelines to identify randomized studies reporting comparative outcomes of neurectomy versus ilioinguinal nerve preservation surgery during Lichtenstein hernia repairs. Studies were identified by searching in PubMed, Scopus, and Web of Science from April 2020. The protocol for this systematic review and meta-analysis was submitted and accepted from PROSPERO: CRD420201610.In this systematic review and meta-analysis, 16 RCTs were included and 1550 patients were evaluated: 756 patients underwent neurectomy (neurectomy group) vs 794 patients underwent ilioinguinal nerve preservation surgery (nerve preservation group). All included studies analyzed Lichtenstein hernia repair. The majority of the new studies and data comes from a relatively narrow geographic region; other bias of this meta-analysis is the suitability of pooling data for many of these studies.P < 0.00001)]. Neurectomy did not significantly increase the groin paresthesia 6 months after surgery at 8.5% (30/353) in the neurectomy group versus 4.5% (17/373) in the nerve preservation group . At 12 months after surgery, there is no advantage of neurectomy over chronic groin pain; no significant differences were found in the 12-month postoperative groin pain rate at 9% (9/100) in the neurectomy group versus 17.85% (20/112) in the inguinal nerve preservation group . One study (115 patients) reported data about paresthesia at 12 months after surgery  and results were not significantly different between the two groups . The subgroup analysis of the studies that identified the IIN showed a significant reduction of the 6th month evaluation of pain in both groups and confirmed the same trend in favor of neurectomy reported in the previous overall analysis: statistically significant reduction of pain 6 months after surgery at 3.79% (6/158) in the neurectomy group versus 14.6% (26/178) in the nerve preservation group .A statistically significant percentage of patients with prosthetic inguinal hernia repair had reduced groin pain at 6 months after surgery at 8.94% (38/425) in the neurectomy group versus 25.11% (113/450) in the nerve preservation group [relative risk (RR) 0.39, 95% confidence interval (CI) 0.28–0.54; Z = 5.60 (Ilioinguinal nerve identification in Lichtenstein inguinal hernia repair is the fundamental step to reduce or avoid postoperative pain. Prophylactic ilioinguinal nerve neurectomy seems to offer some advantages concerning pain in the first 6th month postoperative period, although it might be possible that the small number of cases contributed to the insignificancy regarding paresthesia and hypoesthesia.Nowadays, prudent surgeons should discuss with patients and their families the uncertain benefits and the potential risks of neurectomy before performing the hernioplasty. Inguinal hernia is one of the most common male diseases worldwide . InguinaIlioinguinal nerve section (neurectomy) has been proposed to reduce the incidence of chronic groin pain after inguinal hernia repair . Thus, tWe carried out a systematic literature search  using PubMed, Scopus, and Web of Science. The protocol for this systematic review and meta-analysis was submitted and accepted from PROSPERO: CRD42020161015. The search terms used were inguinal nerve AND hernia, neurectomy AND hernia, ilio-inguinal nerve AND hernia, ilio-inguinal nerve AND neurectomy, and hernia repair AND nerves. Additional search was performed on gray literature on Google Scholar (https://scholar.google.com/) and Google Books (https://books.google.it/). This systematic review of literature was conducted according to the preferred reporting Items for systematic reviews and meta-analyses (PRISMA) guidelines  and the All titles and abstracts were evaluated to identify those that could be included in the analysis. The inclusion criteria were as follows: studies including patients undergoing an inguinal hernia repair with mesh according to the Lichtenstein technique , 20, comThe exclusion criteria include reviews, meta-analysis, clinical cases, editorial opinion articles, and non-RCTs.Postoperative groin pain was assessed according to its severity using a four-point scale and assigning numerical values from 0 to 3 .Among the authors, R.C. and M.S., respectively, handled data extraction and bias risk analysis, subsequently comparing and expressing results based on common opinion.The meta-analysis was carried out including RCTs, which compared neurectomy and ilioinguinal nerve preservation surgery, during Lichtenstein inguinal hernia repair with mesh.For data evaluation and analysis in the included studies, binomial aggregate prevalence estimates were defined and 95% confidence intervals (CI) were calculated using the Review Manager software (RevMan) . Random-Evaluation of the quality of the study. The methodological quality of the studies, included in the meta-analysis, was performed using the Cochrane risk-of-bias assessment tool for RCTs .The search strategy identified 1919 studies, and 70 additional records were identified through other sources . After deduplication, 852 citations were screened of which 821 were excluded on the basis of title and abstract. For the remaining 31 studies, the full texts were obtained and reviewed. Sixteen studies were included –43, and The 16 RCTs included evaluated 1550 patients (Table Most of these studies were performed in Asia (12 RCTs), and only few studies were performed in Europe (2 RCTs) and Africa (2 RCTs). No study was performed in America and Australia. India published the highest number of studies (7 RCTs). Only one study was multicentric. The studies were conducted from 2003 to 2015 and published from 2006 to 2019.Risk of bias in included studies. For details on the risk of bias of the included trials, please see the characteristics of included studies in Supplemental Fig. 1a and Supplemental Fig. 1b and our risk-of-bias criteria in Supplemental Table 1 . If there was no information about the analyzed risk, we left empty spaces in the risk-of-bias table as fourth judgment for the studies).Randomization. Random sequence generation was well explained in nine trials , 41, 43;Allocation. Of the 16 RCTs, four reported details about allocation concealment, and they were considered to have low risk of bias , 36, 42;Blinding of participant and personnel. Of the 16 RCTs, seven reported details about blinding of participants and personnel. Since the studies focused on the surgical procedure, we considered that complete blinding of the personnel was impossible; therefore, we decided to only assess the blinding of the patients. These studies were considered to have low risk of bias , 39, 42.Blinding of outcome assessment. Of the 16 RCTs, eight reported details about blinding of outcome assessment, and they were considered to have low risk of bias , 39, 42.Eight trials did not report information about the blinding of assessment, so they were considered to have unknown blinding information , 40, 41.Incomplete outcome data. We decided to judge RCTs to have low risk of bias if outcome data were missing or missing outcome data were less than 15%, with reasons for missing outcome data unlikely to be related to the true outcome. Consequently, 13 RCTs were judged as having low risk of bias , 41–43.Selective reporting. One trial had no information about secondary outcomes, other than the absence of information about the protocol, marking this item empty . All remOther potential sources of bias. The Cochrane Community did not approve these last two items, so they were added for analyzing two more aspects that we judged important for the outcome of the studies.*Comorbidities associated with worsening or ambiguity. The criteria for having “high risk” of bias were any of the following criteria, other than the local exclusion criteria: bilateral hernia, complicated hernia .*Procedures that may negatively affect the outcome. All studies that clearly identified the ilioinguinal nerve were judged to have “low risk” , 39, 41,P < 0.00001)] and the heterogeneity was very low . The subgroup analysis shows that only mild and moderate chronic groin pains were significantly present in the neurectomy group: mild pain , moderate pain , and severe pain  ; I2 = 0%].The incidence of postoperative groin pain at rest was significantly low in the neurectomy group  . In this analysis, the heterogeneity between the included studies was absent .The incidence of postoperative groin pain when performing daily activities was significantly low in the neurectomy group  . In this analysis, the heterogeneity between the included studies was absent .The incidence of postoperative groin pain after moderate activities was significantly low in the neurectomy group  . In this analysis, the heterogeneity between the included studies was absent .Postoperative groin pain after vigorous activities was significantly low in the neurectomy group  . In this analysis, the heterogeneity between the included studies was absent ; the heterogeneity between the included studies was absent Tau2 = 0.00; Chi2 = 0.39, df = 3(P = 0.94); I2 = 0%  investigated the prevalence of postoperative groin pain 12 months after the intervention (100 neurectomy vs. 112 nerve preservation). These studies revealed no significant differences in the 12-month postoperative groin pain rate: 9% (9/100) in the neurectomy group vs. 17.85% (20/112) in the nerve preservation group  ; the heterogeneity was absent . The subgroup analysis of the different severities of chronic groin pain was not carried out since no study reported data necessary for inclusion. The ilioinguinal nerve was identified in both studies; therefore, we do not report a different subgroup analysis. However, only one study carried out a more accurate analysis of postoperative chronic groin pain; this assessment was performed accounting how the groin pain manifested itself in relation to various physical activities. Regarding the occurrence of pain related to physical activities, no significant differences were noted between the two groups in terms of postoperative groin pain at rest , during daily activities , and after vigorous activities .Only two studies , 32 (n =n = 726) reported 8.5% rate of groin paresthesia at 6 months after surgery (30/353) in neurectomy group vs. 4.5% patients in the nerve preservation group (17/373). This result is positive for inguinal nerve preservation, but it is not statistically significant  ; I2 = 0]. The subgroup analysis of the studies .Eight studies –39, 41  reported the presence of paresthesia 12 months after the intervention . The incidence of paresthesia was lower in preservation group , but it was not statistically significant.Only one study  ; I2 = 30%].In total, seven studies –37, 39 ; the hetP = 0.44)]. However, the heterogeneity was very high .The subgroup analysis of the studies , 36, 39 Only one study   . The subFurthermore, only ten studies reported postoperative pain at 6 months and in three of those pain severity was not evaluated. Only two studies reported postoperative pain at 12 months, with just over 100 patients in each group. In reporting pain outcomes, the high degree of heterogeneity present limits the ability to draw conclusions from the aggregated data. Prior meta-analyses have questioned the suitability of pooling data for many of these studies , 60, 61.Three prior meta-analyses of RCTs on ilioinguinal neurectomy during Lichtenstein repair have been published , 60, 61,P = 0.61)]. Furthermore, the presence of hypoesthesia 6 months after the intervention was lower in inguinal nerve preservation group than neurectomy group, but no statistically significant difference was found . The bias of this results is the lower number of studies included in these analyses , as well as the heterogeneous evaluation of these data for the absence of a common standardized system. The improved use of Dermatome Mapping Test in common clinical practice can represent an adequate system for the standardization of future RCTs [After neurectomy, sensitivity disturbances may accompany pain or may onset separately  In our sure RCTs . In the th month postoperative pain: in effect in both groups with nerve identification a significant reduction of the pain and a trend in favor of neurectomy group was reported.Regardless of surgical choices, the nerve identification is recommended to reduce 6In the context of the previously reported limitations, prophylactic ilioinguinal nerve neurectomy in Lichtenstein hernia repair seems to offer some advantages about pain in the first postoperative period. Considering paresthesia and hypoesthesia, the result was not significant, although it was mostly in favor of preservation; it might be possible that the small number of cases led to this insignificancy.Nowadays, prudent surgeons should discuss with patients and their families the uncertain benefits and the potential risks of neurectomy before performing the hernioplasty.Further research must be conducted, especially in the long-term period, to provide additional data that can confirm the results of this systematic review and meta-analysis. So far, we believe that in the Lichtenstein procedure, prophylactic neurectomy of the ilioinguinal nerve can be a valid choice to reduce the incidence of postoperative chronic pain.Supplementary file1 (DOCX 170 kb)Supplementary file2 (DOCX 28 kb)Supplementary file3 (DOCX 22 kb)Supplementary file4 (DOCX 53 kb)Supplementary file5 (DOCX 44 kb)Supplementary file6 (DOCX 49 kb)Supplementary file7 (DOCX 49 kb)Supplementary file8 (DOCX 43 kb)Supplementary file9 (DOCX 40 kb)Supplementary file10 (DOCX 40 kb)Supplementary file11 (DOCX 40 kb)