Centratherum anthelminticum seed extract (CA) against PCOS in rodents as it is traditionally used to treat diabetes, inflammation, and gynecological problems. The CA was chemically characterized by high-performance liquid chromatography-diode array detection (HPLC-DAD). For the induction of PCOS, a high-fat diet (HFD) was given to all female Wistar rats for nine weeks except the normal control group, which was given a normal chow diet. Estradiol valerate was given to all rats except normal control. After the induction of PCOS, oral metformin (300 mg/kg) was given to the standard group, while CA was orally administered to diseased rats at 250, 500, and 750 mg/kg/day for 28 days. HPLC-DAD analysis revealed that kaempferol-3-pcoumaroylglucoside was present in the highest amount (146.8 ± 1.8 mg/g) of the extract followed by ferulic acid and malvidin-3-(6-caffeoyl)-glucoside. The in vivo results revealed a marked reduction in cholesterol and triglyceride levels in CA treatment groups. A significant rise was observed in progesterone and follicle stimulating hormone with a decrease in luteinizing hormone in the treatment groups as compared to disease control, which indicated normalization of the estrus cycle. The decrease in insulin resistance was characterized by low serum insulin levels in treatment groups. Treatment with CA also reduced inflammatory markers, such as IL-6 and NF-κB in PCOS rats. NrF2 and oxidative stress markers such as catalase, superoxide dismutase, malondialdehyde, and reduced glutathione were also improved by CA in the ovary of diseased rats. Histopathological examination showed the different developmental stages of normal follicles in CA-treated diseased rats which were indicative of a normal fertile estrous cycle. Overall, the results confirmed the efficacy of CA against PCOS in treating estradiol-HFD-induced PCOS due to its antidiabetic, anti-inflammatory, antihyperlipidemic, and antioxidant properties.Polycystic ovary syndrome (PCOS) in females is an endocrine pathological condition of reproductive age which is usually caused by insulin resistance, hyperlipidemia, and oxidative stress. This research was aimed at evaluating the therapeutic effect of the  Polycystic ovary syndrome (PCOS), a reproductive age endocrine disorder, affects 5.6–21.3% of the female population among all ethnicities . The keyAbnormally elevated levels of androstenedione, insulin, luteinizing hormone (LH), and serum testosterone are evident in PCOS patients , 11. HypCentratherum anthelminticum (CA) Kuntze (bitter cumin/black cumin) is a member of the Asteraceae family and is commonly used in Ayurvedic medicine. Over 120 chemical compounds have been discovered in different plant parts of CA. It is rich in phytochemicals, particularly butin, tetrahydroxy chalcone, and tetrahydroxy flavone [Multiple studies in rodent models of PCOS demonstrated that several herbs might be beneficial for the treatment of laboratory and clinical symptoms of PCOS, such as insulin resistance, menstrual disturbances, infertility, hormonal status, and anthropometric indices. These herbs and their extracts can be either used alone or in combination with standard treatments . Centrat flavone . Ayurved flavone .Previous studies demonstrated that the CA had exhibited a reduction in reactive oxygen species (ROS) and lipid peroxidation and improved the antioxidant enzyme activities, including superoxide dismutase (SOD) and catalase (CAT) to protect the damage to macromolecules such as DNA, membrane lipids, and proteins. Moreover, CA caused a significant decrease in both the cardiac and liver function biomarkers associated with intracellular microvesicular steatosis and lipid accumulation . Multipl2O2), sodium chloride (NaCl), chloroform (CHCl3), sodium hydroxide (NaOH), sodium phosphate buffer solution (PBS), pyrogallol, potassium phosphate (K3PO4), trichloroacetic acid (TCA), thiobarbituric acid (TBA), hydrochloric acid (HCl), monosodium phosphate, disodium phosphate, dithiobis nitrobenzoic acid (DTNB), and sodium potassium tartrate were obtained from Sigma-Aldrich, USA. HPLC-DAD , double beam spectrophotometer (Shimadzu UV-1601 Japan), Glucometer AccuChek® Performa (Roche Diabetes Care), and rotary evaporator (RE-501-Germany) were used in the study.Estradiol valerate , hydrogen peroxide  was kept in the refrigerator at 2–8°C till further use.The plant was collected from Punjab and identified by a taxonomist at the University of Punjab (Ref. No. LAH#61120B). Dried seeds of μL of the sample solution was injected and analyzed by HPLC-DAD, according to the previous method [A solution of 100 mg extract/10 ml of methanol was prepared and filtered through a syringe filter into the HPLC vials (2 mL). The injection volume was 50 s method . The C18Thirty-six nulliparous female Wistar rats of weight ranging from 120 to 150 g were obtained and kept at the animal house of Riphah International University. All rats were kept at 25 ± 3°C and 55–65% humidity during acclimatization and experimental study. All protocols were executed as per the guidelines of the Institutional Animal Ethical Committee (Ref. No. REC/RIPS-LHR/2022/058).th week by removing their ovaries for histological examination and compared with that of NCG [High-fat diet (HFD) comprising 40% fat, 20% protein, 36% carbohydrate, and 4% others  was given to all animals for nine weeks, except normal control (NCG) which received normal rodent chow diet , 22. To t of NCG .Rats on a normal chow diet served as NCG. All animals except NCG received HFD and estradiol valerate and were divided into five groups. Disease control (DCG) received distilled water, while animals in the standard therapy group (STG) orally received metformin 300 mg/kg/day . The remth day. For performing OGTT, 75 mg/ml glucose solution was administered via oral route to each animal and blood glucose level was checked at 0, 30, 60, 90, 120, and 180 min after glucose administration. Triglycerides and total cholesterol were estimated by using ELISA kits. Moreover, the blood sample was pulled out after 28 days of therapy by cardiac puncture under anesthesia with chloroform. Blood was collected in ethylenediaminetetraacetic acid (EDTA) tubes. Serum was collected by centrifugation of blood at 1000 rpm and stored in a refrigerator for further use. Liver function tests i.e., glutamic-pyruvic transaminase (SGPT) and glutamic oxaloacetic transaminase (SGOT) were analyzed by standard methods [The blood was taken from the rat tail for an oral glucose tolerance test (OGTT) on the 28 methods .μm were prepared with a microtome. The excised ovarian tissues were stained with hematoxylin and eosin for histological examination under a light microscope [For histopathological studies, the cervical dislocation technique was used to kill the anesthetized animals and ovaries were dissected out through abdominal excision and placed in 10% buffered formaldehyde. The ovarian tissues were fixed in paraffin wax and sections of 4-5 croscope .κB) by PerkinElmer Health Sciences, Inc., USA, were utilized in the experimental study for rats [The ELISA kits of FSH, LH, progesterone, nuclear factor erythroid 2-related factor 2 (NrF2), interleukin (IL-6), and nuclear factor kappa B . The ovarian tissues were thoroughly crushed with a homogenizer. The PBS was added to the crushed tissues to make 10% w/v homogenate. Afterward, the mixtures were centrifuged for 5 min at 1000 rpm, and supernatants were collected to determine oxidative stress parameters .2O2. Absorbance change was measured for 30 S at a wavelength of 240 nm. The CAT activity was stated in U/mg of protein. Indeed, one CAT activity unit was equal to 1 M of hydrogen peroxide decomposed per min at 25°C [CAT activity was estimated by a previously established method. A cuvette was filled with 1 ml supernatant which already contained 1.95 ml phosphate buffer  and 1 ml 30 mM H at 25°C .The MDA is a lipid peroxidation index that is determined by the double heating method. This method is based upon the measurement of absorbance of purple color produced by the TBA reaction. To carry out this estimation, 2.5 ml TCA was mixed with 0.5 ml supernatant. Then, these solutions were kept in a boiling water bath for 15 min followed by cooling down to room temperature. Centrifugation was carried out at 1000 rpm for at least 10 min. Collected supernatant (2 ml) was admixed with 1 ml TBA solution . These solutions were again boiled over a water bath for 15 min. The absorbance was calculated at 532 nm after cooling .µl pyrogallol solution (2.6 mM in 10 mM HCl), and 100 µl sample supernatant. Absorbance was recorded repeatedly at 325 nm after every 30 S. Under the assay conditions, each unit of SOD was equal to the quantity of enzyme needed to achieve 50% pyrogallol autoxidation inhibition [Free radical scavenging capacity in the tissue homogenates was measured. These radicals were produced by pyrogallol auto-oxidation. The reaction mixture was comprised of 2.8 ml PBS, 100 For GSH estimation, a tissue homogenate supernatant of 1 ml was mixed up with an equal volume of 10% TCA. To this mixture, 1 ml phosphate solution was added along with 0.5 ml DTNB reagent was added. Absorbance was taken at 412 nm. The values were stated as nm of GSH per mg of protein .p < 0.01, while these were considered highly significant when p < 0.001.A one-way analysis of variance (ANOVA) test was applied to the data regarding hormonal, lipid and liver parameters, insulin, HbA1c, oxidative stress, and inflammatory parameters using GraphPad Prism 5 . Changes in body weight were assessed by two-way ANOVA. Moreover, the statistically significant data were subjected to Tukey's post hoc test. The results were moderately significant when HPLC-DAD analysis revealed that kaempferol-3-pcoumaroylglucoside was present in the highest amount (146.8 ± 1.8 mg/g) of the extract followed by ferulic acid (145.8 ± 1.21 mg/g) and malvidin-3-(6-caffeoyl)-glucoside (77.1 ± 0.4 mg/g). The detailed phytochemical analysis of the CA extract is shown in Blood glucose level via OGTT was recorded in all groups at the end of the treatment which revealed that the STG and all different doses of CA decreased the blood glucose levels 90 min after glucose administration. The DCG demonstrated the highest rise in blood glucose. The effect of CA 250 mg/kg on blood glucose levels was minimum among all therapeutic groups. The effect of CA on blood glucose levels on OGTT in PCOS rats is shown in p < 0.0001) in insulin as compared to DCG. The effect of treatment with different doses of CA on the serum insulin of PCOS rats is shown in Administration of estradiol and HFD led to a significant increase in insulin level (hyperinsulinemia) of PCOS animals as compared to NCG. The PCOS rats treated with STG and different doses of CA demonstrated a significant decrease  in ovaries as compared to the DCG. Although, all PCOS animals treated with CA showed a significant increase in CAT activity as compared to DCG; however, the CA 500 and 750 mg/kg exhibited a higher level of CAT as compared to the lowest dose of CA. The effect of treatment with different doses of CA on CAT activity in the ovarian tissue of PCOS rats is shown in It was observed that the activity of CAT in the ovary tissue homogenate of PCOS rats had reduced significantly as compared to NCG. In the STG, there was a significant increase in CAT activity  and antral follicles (AF) were observed. Changes in numbers of AF were also observed in the CA 250 mg/kg treated group, along with shrinkage of ovary tissues and decreased number of corpus luteum. Primary and secondary follicles were also seen. However, the most pronounced changes were observed in PCOS rats treated with CA 750 mg/kg group which demonstrated several follicles such as graafian follicle (GF), AF, and PAF at different developmental stages. Corpus luteum was also observed with decreased number of secondary follicles, thus exhibiting the restoration of fertility as shown in Like other endocrine diseases, PCOS is treated by lifestyle modifications and pharmacological and hormonal therapy. Metformin has established its role in the management of PCOS over time, but due to its deleterious effects, pharmacological interventions with multiple herbal extracts have been assessed which showed efficacy alone or in combination with conventional therapy against PCOS . In currDifferent phytochemicals including kaempferol, ferulic acid, malvidin, caffeoylquinic acid, and quercetin derivatives in the plant extract were detected. These components extracted from the plants have different pharmacological effects, including anti-inflammatory, antipyretic, anthelmintic, antioxidant, antimicrobial, antiviral, anticancer, antifilarial, wound healing, and diuretic activity , 33.α-amylase and α-glucosidase, which decrease glucose absorption in the gastrointestinal tract [Insulin plays a vital role in stimulating stromal androgen and ovarian thecal secretion. Insulin resistance, in the form of hyperinsulinemia, induces the overproduction of ovarian androgens. Moreover, terminal differentiation and anovulation are induced by the impact of insulin-like growth factor (IGF)-1 on LH. The IGF-1 enhances the production of estrogen through granulosa cells and acts in regulating aromatase concentration of granulosa cells via synergism with LH and FSH , 35. Insal tract .Steroidal hormones play a pivotal role in sexual maturation and differentiation and are synthesized from cholesterol under the influence of gonadotrophins. These steroidal hormones are produced in steroidogenic tissues involving six different cytochrome P450 enzymes . These gß-hydroxy ß-methylglutaryl CoA reductase (HMG-CoA), the enzyme important for rate-limiting step in the biosynthesis of cholesterol, or an increase in the low-density lipoprotein (LDL-c) breakdown through its receptors on the liver. The decrease in triglycerides may be related to the augmented activity of the endothelial-bound lipoprotein lipase, which leads to the hydrolysis of triglycerides into fatty acids [C. anthelminticum have been established in multiple studies. Previously, it is demonstrated that C. anthelminticum had improved the oxidative stress and lipid profile in triton X-100-induced hyperlipidemic rabbits [The present study showed that the CA had resulted in a reduction of cholesterol and triglyceride level in PCOS rats. The effect of CA on cholesterol might be either due to the inhibition of the ty acids . Hyperli rabbits .The administration of HFD and estradiol influenced the hormonal levels. The effect of estradiol and HFD was evident on the liver as diseased rats showed a significant rise in levels of SGPT and SGOT. This study has shown the ability of CA to regulate the hormonal imbalance caused by the endocrine disorder which had caused an increase in LH and a decrease in progesterone and FSH. It demonstrated the ability of CA to prevent the progression of PCOS.Oxidative stress induced due to hyperlipidemia is one of the associating factors that point to long-term complications of PCOS by enhancing free radical production and suppressing the antioxidant enzyme system in the body . The oxiIt is found that the reduction of SOD and CAT activities and increase in MDA level are the results of oxidative imbalance in reproductive tissues. Oxidative stress in ovarian tissues of PCOS rats is evident in previous studies. Long-term oxidative stress leads to sperm immobility and damage to DNA, enzymes, and lipid peroxidation –39. Therα, IL-6, NF-κB, and IL-1ß are associated with PCOS [κB, NrF2, and IL-6 in PCOS rats which resulted in a reduced risk of chronic inflammation.Multiple shreds of evidence supported that high levels of proinflammatory markers TNFith PCOS . An elevith PCOS . PCOS woith PCOS . This stHistopathological substantiation in the current study showed that the ovaries of HFD and estradiol valerate-treated rats exhibited numerous cysts along with either a very lean or no granulosa layer. Moreover, diseased animals showed anovulation which was indicated by the absence of the corpus luteum. Treatment with CA led to the cyst disappearance and emergence of corpora lutea and normal follicles. Sections from CA 250 mg/kg treatment showed follicles bigger in size and lesser corpora luteum. CA at 759 mg/kg showed several corpus luteum and antral follicles with clearly differentiate oocytes, granulosa cell layer, corona radiate, cumulus oophores, and thecal cells indicating improvement of PCOS .C. anthelminticum displayed an ameliorating effect against PCOS in rats due to anti-inflammatory, antioxidant, antihyperlipidemic, and antidiabetic activities. The plant extract was the most efficacious at 750 mg/kg. These in vivo activities were most probably due to the presence of flavonoids and phenolic compounds which decreased the oxidative stress, insulin resistance, and HbA1c and normalized the hormonal imbalance to restore ovulation.Based on the current study, it can be concluded that the extract of It is suggested that the plant extract must be evaluated for its antiobesogenic potential in diabetic rats. Furthermore, a comprehensive phytochemical analysis and bioassay-based isolation of the multiple phytochemicals responsible for these activities must be carried out to discover a safer, efficacious, and economical treatment of PCOS.


However, understanding the functional and structural deficits at NMJs prior to this loss is crucial for therapeutic strategy design. Should early interventions focus on reversing denervation, or supporting largely intact NMJs that are functionally impaired? We therefore determined when functional and structural deficits appeared in diaphragmatic NMJs relative to the onset of hindlimb tremor (the first overt motor symptoms) We employed electrophysiological recording of NMJ postsynaptic potentials for spontaneous and nerve stimulation-evoked responses. This was correlated with fluorescent imaging microscopy of the postsynaptic acetylcholine receptor (AChR) distribution throughout the postnatal developmental timecourse from 2 weeks to early symptomatic ages.in vivo motor symptoms. Despite this, fluorescent α-bungarotoxin labelling revealed no systematic, progressive changes in 11 comprehensive NMJ morphological parameters  with disease progression. Rather, while NMJs were largely normally-shaped, from 16 weeks there was a progressive and substantial disruption in AChR concentration and distribution within the NMJ footprint.Significant reduction in the amplitudes of spontaneous miniature endplate potentials (mEPPs) and evoked EPPs emerged only at early symptomatic ages . Reductions in mEPP frequency, number of vesicles per EPP, and EPP rise time were seen earlier, at 16weeks, but this reversed by early symptomatic ages. However, the earliest and most striking impairment was an inability to maintain EPP amplitude during a 20 Hz stimulus train, which appeared 6 weeks before overt in vivo, while structural deficits occur 4 weeks later, and predominantly within NMJs. These data suggest initial therapies focused on rectifying suboptimal NMJ function could produce effective relief of symptoms of weakness.Thus, NMJ functional deficits appear at least 6 weeks before motor symptoms  We suggest the appearance of motor symptoms in the mouse model is likely to be equivalent to the point at which most patients would present for diagnosis and treatment. Because the major cause of death is respiratory failure  is the loss of functional neuromuscular junctions (NMJs), producing the weakness and paralysis, and ultimate respiratory arrest, characteristic of the disease. Since 95% of all ALS occurs spontaneously, prophylactic treatments are not likely to be applicable to most patients. Thus, treatment will likely start when symptoms first appear, at least for the foreseeable future and for most sufferers. To design optimal treatments after onset of disease symptoms, it is important to know the condition of a patient’s NMJs at that stage, i.e., to understand what precise issues need to be addressed by the therapy/therapies. For example, whether the earliest symptoms are due largely to the complete removal of NMJs, or more the result of generally impaired function in sick but still viable NMJs. Further, if the latter, it is important to understand how much structural disruption drives the loss of function, or whether the functional loss occurs prior to large-scale morphological disruption. These issues are important for informing the design of treatment therapies and evaluating their likelihood of success. For example, whether the treatment should focus on re-innervation of NMJs that have been withdrawn or enhancing function in NMJs that are still present but functionally impaired. It would seem intrinsically easier and potentially offer hope of more effective symptomatic relief to enhance function in NMJs that are still partly intact, rather than a need to entirely re-establish NMJs that have been completely withdrawn. Finally, if the NMJs are disrupted, it is important to understand if this is secondary to impaired pre-synaptic function, or if postsynaptic structural disruption prevents effective transmission. Again, this will inform the search for the location of the most suitable therapeutic target. While there are many previous studies of NMJs, in both structure and function, in ALS disease models , most lo in life . Therefo failure , the maiInformation on the detailed time course of denervation and associated functional changes specifically in the diaphragm in hSOD1-G93A models of ALS is lacking as most studies examine only at particular, strategic timepoints . MoreoveIn the present study, function was determined by sharp-electrode postsynaptic recording of spontaneous and evoked release of neurotransmitter, quantal analysis of release and ability to maintain release during functionally relevant trains of stimuli (2 min at 20 Hz) . In the 2.2.1.The scoring system used to assess the severity of ALS in the hSOD1-G93A mouse was that designed by 4 = normal, no sign of motor dysfunction3 = hind limb tremors evident when raised by the tail2 = gait abnormalities1 = dragging of at least one hind limb0 = inability to right itself within 30 s after being placed on its back.The oldest animals used in the present study were stage 3, which we termed ‘early symptomatic’, and in our colony were aged 18–22 weeks of age (126–154 days). As this is the earliest detected sign of motor abnormality, we suggest this probably corresponds to the earliest stage at which patients present for treatment.2.2.2.2.1.ad libitum access to standard chow and drinking water). All mice were checked daily. ARRIVE guidelines were followed except that blinding and full randomization were not possible. The HO license required all early symptomatic animals to be used within 24 h of hindleg tremor being detected. Thus, experiments on symptomatic hSOD1-G93A mice occurred within 24 h of animal facility notification after their daily checks. Pseudorandomization of age and genotype occurred by default, as animal usage was determined by small litter size , date of birth/usage, only 1 animal (2 hemidiaphragms) maximum per day, experimentation only on weekdays, age-and littermate matching, and within the ±2 days of age required, and equalizing age group sizes. hSOD1-G93A mice founders were male B6SJL-TgN (SOD1-G93A)1Gur/J (SOD1G93a) from the University of St Andrews colony, that were established and maintained on a congenic C57bl/6 background strain from the University of Aberdeen colony. hSOD1-G93A and wildtype mice from the same litter  license to GSB. Animals were kept in standard husbandry conditions within HO guidelines . The diaphragm was split into two hemi-diaphragm/nerve preparations down the midline, and pinned out on PDMS  lined 35 mm diameter tissue culture dish at room temperature under gassed  physiological saline solution containing (mM): 138.8 NaCl, 4 KCl, 12 NaHCO glucose . Dissect glucose .2.2.2.The nerve was taken into the suction electrode and stimulated with a gradually increasing voltage  using a Master 8 pulse pattern generator delivered through an Isoflex stimulus isolation unit . The health of the preparation was determined by the minimum stimulus to evoke a twitch (<1 V) and to produce the maximum visually assessed twitch (2.4–5 V). For stable electrophysiological recordings from NMJs, nerve-evoked muscle contraction was then blocked by bath application with at least 2 μM μ-conotoxin GIIIB  for 55 min, which was the minimum concentration and duration to achieve total paralysis in healthy adult preparations in response to a nerve stimulation intensity of double the previously determined maximum. The toxin exposure was extended, and then if necessary applied at a higher concentration, for muscles from mice of both genotypes at 2–4 weeks and also at early symptomatic stages in hSOD1-G93A, up to a maximum of 8 μM and 120 min. All muscles were tested at the shortest time and lowest concentration (2 μM for 55 min) before toxin exposure was increased if necessary. Once paralysis was established, toxin was removed to avoid nerve block with excessive incubation.μ-Conotoxin GIIIB is a highly selective and high affinity antagonist for rodent mammalian skeletal muscle voltage-gated sodium channels  with no 2.2.3.A sharp glass intracellular microelectrode filled with 3 M KCl was used to record spontaneous miniature end plate potentials (mEPPs), evoked end plate potentials (EPPs) and resting membrane potential (RMP). Electrophysiological recordings used an Axoclamp 2B amplifier  and were stored onto a computer hard drive using Strathclyde University Electrophysiology Software Whole Cell Program . Electrode placement was controlled visually under a modified Nikon Optiphot compound microscope  using a 3.5x Zeiss 3.2/0.07  objective and a free-standing stage. NMJs were located by impaling near one of the first small branches leaving the main nerve trunk as it enters the muscle, with a glass microelectrode. NMJs were identified by the presence of mEPPs, plus an EPP rise time of ≤1.5 ms, indicating the recording electrode is within 200 μm of the NMJ . mEPPs w2.2.4.In some muscles. After recording from 10 NMJs, responses to 20 Hz were recorded. A further NMJ impalement was made and the first 30 s of mEPP and 3x EPPs at 0.1 Hz were again recorded. Then, to examine the ability of the terminals to maintain transmitter release, EPP trains were recorded in response to a continuous stimulus train of 20 Hz for 2 min.2.3.After electrophysiological recording some hemi-diaphragm/phrenic-nerve preparations  initially with a 3.5x  objective to locate the end plate band. High magnification images were acquired through a 40x water emersion objective , with a Prior HBO 100 Hg lamp illuminator with an excitation/emission filter of 510–560/590 nm (Carl Zeiss MicroImaging GmbH), Shutterhub and Acquisitionhub , using a Retiga ER  digital camera and captured onto a PC hard drive, using Volocity 5.3.1 . Twenty 2.4.2.4.1.mEPP and EPP amplitude was measured, and the RMP at which each of these was recorded was noted. This was used in calculations later, as RMP often changed over the duration of the recording at each NMJ. Recordings in which RMP became more positive than −50 mV at any time were rejected. The quantal content of the EPP  was calculated after correcting EPPs for non-linear summation, with the following formula .QC = E / [M x (1 – f (E/Vm))].E = amplitude of EPP.M = mean amplitude of mEPP.Vm = resting membrane potential.f = correction factor for non-linear summation (0.8 was used).To compare mEPP and EPP amplitude between muscle fibers and muscles, they were corrected to -70 mV, using the equation:mEPP) or EPP (70/VEPP), as appropriate.M corrected = mEPP  at which the responses were recorded.V2.4.1.1.p-value of <0.05 was taken as significant.These data are presented as mean ± standard error of the mean (SEM). Analysis of differences between experimental and control groups was carried out using a two-way analysis of variance (ANOVA), followed by individual mean post-tests at specific ages with Bonferroni correction to test for differences between experimental and control groups. A 2.4.2.a. Endplate areab. Endplate diameterc. Endplate perimeterd. Acetylcholine receptor (AChR) areae. AChR perimeter lengthf. Number of AChR clustersFor this analysis, as many NMJs as reached criterion of ‘en face’ (<10% obscured/curved around muscle fiber) were analyzed. The Neuro-morph semi-automated workflow developed by  was usedg. Average AChR cluster area (d/f)h. Endplate area unoccupied by AChRs (a-d)i. Compactness ((a/d)*100)j. Fragmentation (1-(1/f))The further 4 derived variables from these parameters were:The person performing the image analysis was blinded to the genotype of the mice. The accuracy and reproducibility of the semi-automated NMJ-morph algorithm was first verified manually, where the NMJ parameters were compared when determined manually and by different automatic threshold criteria in a sub-set of images from 3 mice at different ages and genotypes. The automatic selection criteria were refined iteratively until manually tracing the NMJ using the drawing tool on ImageJ, produced values <10% different from the semi-automated ‘Huang’ threshold approach. At this point, in fact, the majority of errors were < 5%. Once such minimal discrepancy was routinely achieved between the methods, the NMJ-morph semi-automated algorithm was used to derive the variables of all NMJs  and smoothed to remove sporadic pixel intensity maxima or minima. An intensity frequency histogram was then generated for the entire image, and values plotted in Excel. This generated a curve with a bimodal distribution . The lar2.4.4.1.p < 0.05 was taken as significant.Data are presented as mean ± SEM. Differences in mean ratios were assessed by 2-Way ANOVA comparing age and genotype, with a Bonferroni-corrected post-test between genotypes at specific ages. A 2.4.5.For some muscles at each age, NMJs were analyzed for details of AChR disruption  appearance , sub-div2.4.5.1.p < 0.05 was taken as significant.Many of these data were not normally distributed, so histogram data for incidence of atypical AChR distributions were analyzed by non-parameteric statistical methods, and data are presented as median ± interquartile range. A 3.3.1.p < 0.001, Student’s t-test). Thus, they developed the usual early symptoms, but at a later date. A lower-than-average body mass has been related with ALS in humans, which may be due to hyper-metabolism that develops in ALS  of 18–22 weeks old. Each group was age-matched with wildtype littermates. The parameters, and their potential pathological indication, are given each section.3.2.1.p < 0.0001, Two-way ANOVA), beginning at 1.48 ± 0.09 mV  at 2 weeks and reducing to 1.19 ± 0.05 mV  at 18–22 weeks. This reflects the growth in diameter, and hence decrease in input resistance, of the postsynaptic muscle fiber  of 18–22 weeks old, which were age-matched with wildtype littermates. Typical mEPPs at each age are shown in le fiber .n = 72, eight muscles) compared to WT . At 12 and 16 weeks, mEPP amplitudes are the same as in wildtype mice. However, in early symptomatic hSOD1-G93A mice, mEPP amplitudes had become significantly smaller  than in age-matched wildtype controls , a reduction of 21.0%.In hSOD1-G93A mice, initially there is a trend for mEPP amplitudes to be larger than in wildtype mice of the same age , becomin3.2.2.p < 0.0001, Two-way ANOVA), again consistent with studies in rat muscles .Endplate potentials result from the simultaneous release of many vesicles, which in healthy NMJs is enough to depolarize the muscle fiber to threshold for muscle fiber contraction. It seems likely that an early indicator of disease in motor terminals would be impaired neurotransmitter release, and so decreased EPP amplitude. This would reduce the likelihood of reaching threshold for triggering contraction, which might be a contributory factor to the symptoms of muscle weakness in ALS. Typical EPPs for wildtype and hSOD1-G93A are shown in  muscles , and ref3.2.3.p < 0.001, Two-way ANOVA – Bonferroni post-test). However, despite the decrease in mEPP and EPP amplitudes (EPP amplitude is a function of the number of vesicles (mEPPs) being released per nerve stimulus. This is another important measure of the health and efficacy of a motor nerve terminal. The actual number of vesicles released per action potential  was calculated to better understand the timing and effects of disease progression. In wildtype mice, as in previous rat studies, QC increased with age. In hSOD1-G93A mice, QC was not significantly different from wildtype at most ages  but therplitudes , the QC 3.2.4.p < 0.01, Two-way ANOVA – Bonferroni post-test  can indicate disrupted synchronicity of release, alterations in activity of the AChesterase breaking down ACh in the NMJ synaptic cleft. Reduced (less negative) resting membrane potential can indicate the disease directly affecting the muscle, or inadequate innervation. In wild-type NMJs, mEPP frequency increased up to 12 weeks old when it plateaued. In hSOD1-G93A mice, as with QC, mEPP frequency was normal until 16 weeks, when it was significantly reduced by 43.7%; ost-test  but agaip < 0.05, Two-way ANOVA – Bonferroni post-test; EPP rise time remained effectively constant throughout development in wild-type muscles. Interestingly, in hSOD1-G93A, the EPP rise time was significantly faster at 12 weeks old .RMP in wildtype mice did not change significantly during postnatal development and, at least for muscle fibers with mEPPs and EPPs, RMP in hSOD1-G93A was not significantly different .The ability to maintain EPP amplitude  was compin vivo activity appeared at 12 weeks, which is 6 weeks before obvious early motor symptoms.Thus, overall, NMJ deficits in the ability to maintain release (EPP amplitude rundown) in trains typical of 3.3.The efficacy of neuromuscular transmission depends upon the strong structural correlation between the motor nerve terminal that releases the neurotransmitter and the postsynaptic ACh receptor (AChR) distribution with which the ligand interacts. Given these changes in presynaptic transmitter release, it was therefore important to determine if they were accompanied by changes in the AChR distribution postsynaptically. Thus, the AChR distribution was next examined in these same muscles.Immediately after electrophysiological recording, TRITC-labeled α-bungarotoxin was added to the muscle superfusate to label the AChR distribution at the NMJ. In healthy muscles, this postsynaptic AChR distribution closely reflects that of the overlying presynaptic terminal, although this was not visualized in the current experiments. However, changes to the AChR distribution would still indicate an impact of disease .The postsynaptic morphology was analyzed using the NMJ-morph algorithm , fractal1. No disruption .2. A global change in end-plate area or shape.a. Endplates decrease in size .b. Endplates, at least initially, increase in size .3. Local changes in end-plate shape and AChR distribution.a. Fragmentation .b. Local or global redistribution of AChRs across the whole endplate .While it is known that NMJs become denervated during ALS, and axon retraction bulbs have been demonstrated as the endpoint of that process , the iniThese categories, of course, may each occur but at different times. However, it might be useful clinically to identify a particular characteristic as a biomarker of disease progression, perhaps in motor point biopsies. To try to understand which changes occurred and when, 10 postsynaptic morphological parameters were quantified from 4 weeks to early symptomatic ages: (see Methods for details). Many of these data were not normally distributed, so all were analyzed by non-parameteric statistical methods.Images of typical AChR distributions at NMJs at each age are seen in p < 0.005 at 12, 16 and symptomatic; F-test) and number of AChR clusters .In general, parameters that increased with age were AChR area and perimeter, end-plate area, perimeter and diameter, plus area unoccupied by AChRs. Conversely, this produced reduced compactness and area per AChR cluster with age. The number of clusters and fragmentation was increased, only in the oldest age-group. However, as stated above, none of these showed sustained, statistically significant differences between genotypes, even at symptomatic stages. In contrast, the following parameters did show sustained and significant increases in variability from 12 weeks onwards: AChR area  and early-symptomatic ages . Thus, hSOD1-G93A may reach mature AChR perimeter complexity early , then wild-type NMJs catch up, only for the fractal value to increase disproportionately at early symptomatic stages in hSOD1-G93A mice, when remodeling might be expected to increase.Fractal analysis of the AChR perimeter, might detect an increase in complexity if AChRs are remodeling. Conversely, the AChR area with lowered labeling intensity would indicate withdrawal of AChRs within a stable endplate perimeter. Fractal analysis of the end-plate perimeter  revealedp < 0.0001, wildtype n = 30, hSOD1-G93A n = 45, Bonferroni post-test). This disruption increased with disease progression , whereas it plateaued in the wildtype NMJs from 12 weeks.The most robust indicator of NMJ structural disruption turned out to be the proportion of each NMJ with lower AChR labeling intensity. As shown in Overall, this analysis indicated there was no large-scale disease-driven shrinkage or growth in the footprint of the NMJ. Rather, there was a re-distribution of AChRs within the original footprint. We therefore then undertook a more detailed examination of AChR patterns within the NMJ. In wildtype NMJs, where detail of AChR distributions were seen, this was normally in ‘fingerprint-like’ linear arrays, reflecting concentrations at the crests of postjunctional folds . In NMJsThese two parameters  were quantified in 20 NMJs from each muscle of 2–5 mice at each age (see Methods).Small floccular AChR regions were seen at an essentially constant rate of <5% in wildtype NMJs across all ages . There w4.in vivo motor symptoms emerge, and their extent. Characterizing this could give invaluable insights into how long the underlying processes have been developing and, thereby, indicate what any treatment might need to overcome even at these earliest clinical stages. Finally, correlation of electrophysiology and morphology in the same muscle preparation aimed to indicate the likely contribution, and nature, of structural disruption to the physiological impairment. Overall, therefore, we aimed to understand the processes that might need to be corrected within NMJs that are still functional, although impaired, to offset symptoms. Therefore, this study aimed to give a clear timeline of functional and structural changes, their relative extent and contributions as the context within which therapeutic intervention would occur.This study aimed to understand the physiological and anatomical condition at NMJs when the first, mild motor impairments become detectable in ALS. We used hSOD1-G93A mice, the most widely used model of ALS. By understanding this, we aimed to gain an insight into the level of disruption likely to be present when ALS patients first present to the clinician. In motor terms, for these mice, the proxy here was the appearance of tremor in the hindlimbs when the hind quarters were elevated clear of the gound by the tail. By charting NMJ morphology and neurotransmission throughout postnatal development, this study also aimed to identify how many identified disruptions were present before The hSOD1-G93A mice in our colony had a delayed onset of the condition  relative to original strain . ExactlyIt is also possible that the functional and morphological deficits seen reflect an intrinsic difference in vulnerability to axotomy in the acute experiments used to make the observations, since a number of studies have examined, for example, Wallerian degeneration under similar conditions . Howeverin vivo-like activity of maintained trains of stimulation that much more substantial, and progressive, deficits became apparent. Thus, EPP amplitude during 20 Hz trains of stimuli fell to ~50% of wildtype at the end of 2 min trains from 12 weeks of age, well before the emergence of overt motor symptoms. While we did not seek to determine the cause of this, two pathologies are known to be present that could be responsible. First, a shortage of vesicles available for release, due to a reduced vesicle density  tended to be larger until 8 weeks, in agreement with a previous electrophysiological study of diaphragm NMJs from presymptomatic mice . The amp density  and, sec density . Given tSimilarly, there were no gross changes in morphological parameters at NMJs as determined by NMJ-morph, which aims to provide a robust analysis of the principal components of NMJ morphology. In essence, this analysis shows that the footprint of the AChR distribution changed little even by symptomatic stages, although it must be remembered that this was the earliest symptomatic stage, and there was a significant increase in variability of some parameters. The lack of change in detailed footprint parameters reported in the present study is in agreement with previous less-detailed studies , which fFinally, the percentage of NMJs showing impaired ability to maintain release during trains at early symptomatic stages was very widespread. Although not examined systematically, it was certainly the great majority impaired in this way. That is, most nerve terminals, perhaps all, had functional impairment at early symptomatic stages. In contrast, only ~20% of NMJs had >50% of their area appearing floccular (= denervation-like) appearance, with another ~20% at <50%. If the latter are only partially denervated, this implied that as many as 80% of NMJs may retain some structurally intact innervation and function at this stage.Thus, overall, substantial functional disruption, i.e., an inability to maintain release, occurred 6 weeks before the earliest motor symptoms, followed 4 weeks later by floccular redistribution and reduced expression of AChRs within the NMJs, which likely reflects the onset of denervation. This occurs 2 weeks before overt motor symptoms appeared. There was never a substantial NMJ shape change. Rather, >20% of NMJs developed floccular or granular AChR distributions, and > 50% had developed significant areas of the NMJ with reduced AChR labeling density. These observations indicate, therefore, that the functional changes precede the AChR redistribution and, therefore, the motor terminals are impaired for a significant period before AChR redistribution. It seems likely that at least 80%, and possibly as many as 90%  of NMJs The original contributions presented in the study are included in the article/The animal study was reviewed and approved by University of Aberdeen, Animal Welfare and Ethical Review Board.JM, GB, and GM contributed to conception and design of the study. JM, IM, MD, CG, and RB performed the data analysis and statistical analysis. JM wrote the first draft of the manuscript. GB, GM, IM, MD, CG, and RB wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.This project was funded by a PhD studentship to JM from Motor Neurone Disease Scotland, on grant number RGB3753 to GB and GM.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.https://www.frontiersin.org/articles/10.3389/fnmol.2023.1169075/full#supplementary-materialThe Supplementary material for this article can be found online at: Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.


The equivalent ICURs were €15139 per QALY gained  for 29–31wGA infants and €14719 per QALY gained  for 32–35wGA infants. The model was most sensitive to rates of long-term sequelae, utility scores, palivizumab cost, and palivizumab efficacy. Palivizumab remained cost-effective in all scenario analyses, including a scenario wherein RSVH infants received palivizumab without a reduction in long-term sequelae and experienced a 6-year duration of respiratory morbidity (ICUR: €27948 per QALY gained). In conclusion, palivizumab remains cost-effective versus no prophylaxis in otherwise healthy Italian preterm infants born 29–35wGA. The IRST can help guide cost-effective use of palivizumab in 32–35wGA infants.Since the last Italian cost-utility assessment of palivizumab in 2009, new data on the burden of respiratory syncytial virus (RSV) and an International Risk Scoring Tool (IRST) have become available. The objective of this study was to provide an up-to-date cost-utility assessment of palivizumab versus no prophylaxis for the prevention of severe RSV infection in otherwise healthy Italian infants born at 29–31 weeks’ gestational age (wGA) infants and those 32–35wGA infants categorized as either moderate- or high-risk of RSV-hospitalization (RSVH) by the IRST. A decision tree was constructed in which infants received palivizumab or no prophylaxis and then could experience: i) RSVH; ii) emergency room medically-attended RSV-infection (MARI); or, iii) remain uninfected/non-medically attended. RSVH cases that required intensive care unit admission could die (0.43%). Respiratory morbidity was considered in all surviving infants up to 18 years of age. Hospitalization rates were derived from Italian data combined with efficacy from the IMpact-RSV trial. Palivizumab costs were calculated from vial prices (50mg: €490.37 100mg: €814.34) and Italian birth statistics combined with a growth algorithm. A lifetime horizon and healthcare and societal costs were included. The incremental cost-utility ratio (ICUR) was €14814  Globally, it is estimated that 12.9 million lower respiratory tract infections (LRTI), 2.2 million hospitalizations and >66,000 deaths in children <1 year are attributable to the virus each year [ factors –7. Appro factors .versus no prophylaxis in premature infants without bronchopulmonary dysplasia from the Italian National Health Service perspective (ICUR per QALY gained of €9380 in <33wGA infants and €14937 in 33–35wGA infants) [Passive immunoprophylaxis with palivizumab is currently the only intervention that is widely available to prevent RSV-LRTI in high-risk infants, including premature infants born at ≤35wGA. Nirsevimab will provide an additional option for immunoprophylaxis, but whilst it has been recently approved in several countries, it is not yet available for clinical use. In Italy, a previous health economic analysis published in 2009 demonstrated palivizumab to be cost-effective infants) . Howeverinfants) , 11.versus no prophylaxis in otherwise healthy Italian premature infants born at 29–35wGA, incorporating use of the IRST as well as the latest epidemiological and cost data.Since the publication of the Italian cost-analysis of palivizumab , there hversus no prophylaxis in all 29–31wGA infants and those 32–35wGA infants who are at moderate- and high-risk of RSV-LRTI, as determined by the IRST [This cost-utility analysis assessed the use of palivizumab the IRST . The anathe IRST .To inform the development of the current analysis, a systematic literature review was undertaken to identify the key characteristics of previous cost-effectiveness and cost-utility models . This inA lifetime decision-tree model was developed wherein prophylaxed/non-prophylaxed infants had RSVH, MARI, or remained uninfected/non-medically attended . Followiversus prophylaxed infants being 4.9% versus 1.8% and 10.1% versus 1.8% in 29–31wGA and 32–35wGA infants, respectively. These rates translate to relative risk reductions of 63.3% and 82.2%. The effect of palivizumab on the incidence of respiratory morbidity was calculated by generating a curve of best fit using data from Simoes et al. 2007 [et al. 2013 [et al. 2013 [et al. 2017 [The efficacy of palivizumab was drawn from the Impact-RSV study  the RSVHal. 2007 , Blankenal. 2013  and Yoshal. 2013 , as preval. 2017 .et al. 2019 [th December 2022), with the same duration (mean 6.8 days) applied to both prophylaxed and non-prophylaxed infants. ICU admission rates  were calculated from previous studies [The RSVH rate for 29–31wGA infants not receiving palivizumab was 5.88%, and was abstracted from the study by Priante al. 2019 . The RSVal. 2019  to the n studies , 31. For studies , 33.The IRST was developed using logistic regression analysis of a pooled dataset of 13,475 infants born 32-35wGA drawn from six observational studies . Of the MARI rates were taken from the palivizumab arm of the motavizumab trial  wherein et al. 2017 [versus no prophylaxis for 32–35wGA infants up to 6 years of age. This approach was adopted in the current model for 0–6 years of age and extended to age 18 years using data on respiratory morbidity from the Swedish prospective study by Sigurs et al. [et al. data [et al. data [A previously published algorithm by Sanchez-Luna al. 2017  was usedal. 2017 . The algal. 2017  in combial. 2017 –27 to ess et al. , 39–41. al. data , 39–41 wal. data . This apal. data , 39–41 ial. data .Following RSVH or MARI, any long-term respiratory morbidity was assumed to continue until 18 years of age, although with lower rates for the latter children . In thosper a UK study of premature infants (median 27wGA) with chronic lung disease and RSVH (and a control group without RSVH) until 5 years of age [To account for the impact of RSVH on infants’ quality of life (QoL), a utility value of 0.60 was applied for the duration of the RSVH (6.8 days). This was extracted from a US cost-analysis , which is of age . For nons of age  was usedIn the absence of any RSV-specific utility data on the impact of long-term respiratory morbidity on QoL, a value of 0.79 was adopted. This was based on a study of children with mild-and-moderate asthma symptoms .et al. 2020 [per 29–31wGA infant and €3,422 per 32–35wGA infant. Administration by a nurse was estimated to cost an additional €86.98 per infant based on the cost of a general outpatient appointment [per infant .Costs were calculated on an individual level and reflected the costs of RSVH, MARI, ICU admission, the ongoing costs of respiratory morbidity, and societal costs as well as the acquisition and administration of palivizumab in Italy . All cosal. 2020 . Based oointment  and an aet al. 2018 [To account for fewer births in the 29–31wGA group than in the 32–35wGA, calculations were weighted on the basis that 25.06% of the total cohort of interest would be 29–31wGA infants and 74.94% would be 32–35wGA infants. This was based on Canadian birth statistics over the years 2016–2020, in the absence of publicly available Italian data . The proal. 2018 .per QALY gained (ICUR) when palivizumab was compared to no intervention. A QALY is a summary outcome measure that combines the impact of an intervention in terms of both the quality and quantity of life; a value of 1 indicates a year of perfect health; lower scores represent reduced life expectancy and/or reduced quality of life.As recommended by established guidelines –59, outcet al. [per plotted point was 1000.Commensurate with best practice,  uncertaiet al.  The defaper QLAY gained.Cost-effectiveness acceptability curves (CEACs) were used to represent the uncertainty. The CEAC presents the probability of palivizumab to be cost-effective against no prophylaxis at different willingness to pay thresholds et al. 2019 [Various scenario analyses were undertaken. These included: i) the exclusion of indirect/societal costs ii) removal of discounting; iii) inclusion of vial sharing with 5% wastage ; iv) reduction of the duration of respiratory morbidity to 13 or 6 years; v) standardization of hospitalization rate in non-prophylaxed infants based on data from Priante al. 2019 ; vi) estvia consultation with acknowledged clinical experts in RSV  and comparison with previous cost-analyses of palivizumab identified during the systematic review  compared: number of RSVHs; ICU admittance; and mortality rate. As FLIP-2 considered all 32–35wGA infants, the total population, regardless of risk status, was used for validation purposes.The model structure, data and assumptions were validated c review . Model cThe model was developed using Microsoft Excel 365.per QALY gained when palivizumab was compared with no prophylaxis. When considering 29–31wGA and 32–35wGA infants separately, the corresponding ICURs were €15139 and €14719 per QALY gained, respectively.Palivizumab prophylaxis of all 29–31wGA infants and those 32–35wGA infants identified as moderate- or high-risk for RSVH by the IRST, was associated with increased treatment and indirect costs but led to a reduction in direct healthcare costs and improved QoL compared to no prophylaxis . In the per QALY gained was predicted with palivizumab versus no-prophylaxis for the 29–35wGA group, with 89.9% of PSA runs falling below a €40000 per QALY willingness to pay (WTP) threshold , palivizumab efficacy and average birth weight of 32–35wGA infants , ICU admittance (20% versus 17.9%), and mortality rate (0.003% versus 0%).No marked differences between the model predictions and actual outcomes of FLIP-2  were fouper QALY gained (mean ICUR: €15430 per QALY). This is approximately €25000 below the €40000 per QALY WTP threshold in Italy, as identified by the Italian Health Economics Association [versus €15139 per QALY gained, respectively), illustrating the benefits in the former group of targeting palivizumab use via the IRST. Prophylaxis with palivizumab remained cost-effective versus no intervention in all scenarios examined, and for the high and moderate risk groups when assessed individually  and assumed a higher rate of palivizumab efficacy in reducing RSVH, particularly in the more premature infants (80.5% and 84.5% versus 63.3% and 82.2%). Their reported ICU admission rates were 3% and 1% in non-prophylaxed and prophylaxed infants, respectively, versus 20.0% and 8.7% of those hospitalized in our model [et al. 2019 [et al. 2018 [post hoc analysis of the IMpact-RSV study (published in 2014) that provided age group-specific data and was not available to the authors of the previous analysis [et al. [In the previous Italian analysis  the authur model , while tur model , neitheral. 2018  which deanalysis . Furtheranalysis , 31, incversus 0.27% in the previous Italian model. Of note, our study utilized available Italian data to determine the birthweight  and average number of treatment visits per infant (4.21), while palivizumab cost  was determined based on the amount of palivizumab per injection calculated according to predicted growth. This contrasts to the previous analysis which assumed a standardized palivizumab cost of €3099.84 and 5 injections for all infants [per QALY gained is underscored by its identification as one of the five most important variables in the DSA.Mortality rates in our study were also markedly more conservative than used in the previous Italian analysis, reflecting the increasing recognition that death due to RSV is a rare event in otherwise healthy preterm infants in the modern healthcare setting. Our study assumed a mortality rate of 0.43% and applied it only to those infants admitted to the ICU, whereas the previous Italian analysis  applied Indirect costs were found to be another potentially important consideration in the cost-utility of palivizumab. Our systematic review revealed that many previous analyses, including the one from Italy, adopted the healthcare provider perspective, and therefore excluded societal costs . In thosAnother clinically relevant component that differentiates the current analysis from previous assessments of palivizumab cost-effectiveness was the inclusion of MARI. Although increasingly characterized as a significant contributor to the overall burden of RSV , 19, 20,per QALY gained) and 6 years (ICUR: €27381 per QALY gained). Moreover, a scenario that limited the duration of the respiratory morbidity to 6 years and negated the effect of palivizumab in preventing long-term wheeze in infants with RSVH  demonstrated that the ICUR was still well within the WTP threshold of €40000 per QALY at €27948 per QALY gained.Consideration of the impact of RSV beyond the initial RSV infection and/or hospitalization was addressed differently in our model and the previous Italian one. In the previous model , in commet al. 2004 [et al. [et al. [et al. [per QALY , that the model was validated against outcomes from FLIP-2 [A key limitation of our model was the availability of data describing the short- and long-term QoL outcomes for infants born at 29–35wGA, highlighted by the fact that the DSA identified utility scores as a principal driver of the ICUR. The data utilized in the model were from Greenough al. 2004 , which aal. 2004 , 45, 69.al. 2004 . More re [et al.  and Diez [et al.  included [et al. , which w [et al. . Another [et al. ) and the [et al. –27 in it [et al. , 40, 41) [et al.  are an am FLIP-2 , 72–74.This analysis established that palivizumab prophylaxis of otherwise healthy preterm Italian infants born at 29–35wGA is a cost-effective strategy compared to no prophylaxis. Adoption of the IRST in 32–35wGA infants can help optimize cost-utility and reduce overall costs whilst still protecting most of these infants. Given the mounting cost pressure on healthcare resources, these results re-affirm the role of palivizumab in preventing severe RSV-LRTI and its consequences in otherwise healthy preterm infants and present a practical rationale for its ongoing use.S1 Appendix(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.


Herein, we constructed a near-infrared (NIR)-responsive carrier-free berberine hydrochloride /indocyanine green  nanoparticles (BI NPs) for synergistic antibacterial of an infected wound. Through electrostatic interaction and π–π stacking, the hydrophobic BH and amphiphilic ICG are initially self-assembled to generate carrier-free nanoparticles. The obtained BI NPs demonstrated NIR-responsive drug release behavior and better photothermal conversion efficiency of up to 36%. In addition, BI NPs stimulated by NIR laser exhibited remarkable antibacterial activity, which realized the synergistic antibacterial treatment and promoted infected wound healing. In summary, the current research results provided a candidate strategy for self-assembling new BI NPs to treat bacterial infections synergistically.Bacterial infections cause severe health conditions, resulting in a significant economic burden for the public health system. Although natural phytochemicals are considered promising anti-bacterial agents, they suffer from several limitations, such as poor water solubility and low bioavailability   Bacterial infection poses a significant threat to skin wound healing , which pStaphylococcus aureus (S. aureus), BH exerts an antibacterial effect by inhibiting the activity of glutamine synthetase and transpeptidase affect the synthesis of amino acids and the pathogenicity of virulence factors [in vivo have hindered the broad application of BH.Phytochemicals refer to an extensive range of chemical compounds found in plants. Based on their chemical structure, these phytochemicals are classified into several major categories: alkaloids, flavonoids, terpenoids, polyphenols and sulfur-containing phytochemicals . These p factors . It is w factors . Therefo factors . HoweverIn recent decades, with the continuous improvement in nanotechnology, various drug delivery systems based on nanoplatforms have emerged as an effective approach to overcome the shortcomings of clinical applications. Drug delivery systems could achieve controlled release of drugs at specific sites, enhance the endurance and water solubility of drugs. Researchers came up with several nano-drug delivery systems, such as liposomes , polymerIn recent years, near-infrared (NIR)-responsive drug release systems received considerable attention due to their straightforward operation, simple control and effective tissue penetration. Briefly, once an NIR laser is irradiated, drug release is achieved by the photothermal effect, which refers to the increase in temperature caused by the photosensitizer . Furtherin vitro. Finally, BI NPs were successfully applied to repair the S.aureus-infected skin wounds.Herein, we constructed an NIR-responsive antibacterial platform based on BH/ICG nanoparticles (BI NPs) by direct self-assembly for synergistic chemotherapy/PTT . FirstlyS. aureus (ATCC 29213) and Escherichia coli  were attained from the China Center of Industrial Culture Collection .BH was attained from J&K Scientific . ICG was attained from Sangon Biotech. Co., Ltd. . Dimethyl sulfoxide (DMSO) and agar were attained from Sinopharm United Medical Device Co., Ltd. . Luria-Bertani (LB) was attained from Qingdao Hope Bio-Technology Co., Ltd. . A facile self-assembly approach was used to prepare BI NPs, with minor adjustments based on the reported conditions . FirstlyThe morphology and size of BI NPs were investigated using field emission-scanning electron microscopy  and transmission electron microscopy . Ultraviolet-visible-Near Infrared (UV-vis-NIR) spectrophotometer  was used to measure the UV absorption spectra of the samples, with a scanning range of 200–1100 nm. An attenuated total internal reflectance Fourier-transform infrared (ATR-FTIR) spectrophotometer  was employed to measure the infrared absorption spectra of samples. The surface potential of BI NPs dispersed in deionized water was evaluated using the dynamic light scattering measurements . An X-ray diffraction analyzer  was utilized to determine the crystal structure of the sample.In order to explore the effect of NIR laser on BH release behavior from BI NPs, 1 ml of BI NPs dispersion (1 mg/ml) was incubated in a shaker at 37°C. As for NIR groups, an 808 nm NIR laser was used to irradiate BI NPs dispersion for 3 min. After that, BI NPs dispersion was centrifuged and 1 ml of supernatant was taken at each appointed time , and 1 ml supernatant was taken to measure absorbance value at 360 nm using a UV-vis spectrophotometer. In addition, an equal amount of phosphate buffered saline (PBS) should be added immediately after each removal of the supernatant. The concentrations of BH were calculated against the matching standard curve at each time point. The cumulative release was calculated as described in 2) was used to irradiate 200 μl of water, ICG solution and BI NPs dispersion. Next, the photothermal property of BI NPs was then examined in relation to the impacts of power density and concentration. An NIR thermal imaging instrument  was employed to track all temperature changes every 30 s during the process. The photothermal stability of BI NPs was investigated by repeated irradiation for four times under the same conditions. The photothermal conversion efficiency (η) was calculated according to the previously reported method [To assess the photothermal performance of BI NPs, the photothermal effects of several samples under NIR laser irradiation was initially compared. An 808 nm NIR laser . Then, equal amounts of bacteria suspension  were mixed with different concentrations of BH, ICG solution and BI NPs dispersion, respectively, and constantly shaken at 37°C for 18 h. Before shaking, the NIR groups were additionally exposed to an 808 nm NIR laser (1.0 W/cm2) for 3 min. Subsequently, the treated bacterial samples were then diluted appropriate times and evenly spread over LB agar plates for another 18 h of incubation at 37°C. Finally, the colonies formed were photographed and counted to calculate the inhibition rate.The antibacterial effects of BI NPs on A live/dead bacterial viability kit was utilized to stain the bacterial after treatment with different samples and culture at 37°C for 18 h. Then, a fluorescence microscopy  was utilized to capture the fluorescent images of bacteria.The bacterial-specific morphological alterations were examined using the FE-SEM. The bacterial suspension was treated as described above and fixed with glutaraldehyde (2.5%) for 4–6 h. Next, the bacteria were dehydrated by gradients of 20%, 40%, 60%, 80% and 100% ethanol. Finally, the bacterial suspension after the last dehydration was diluted with ethanol to a suitable concentration and then dropped on the silicon wafer. After naturally drying, the bacteria were observed with FE-SEM.S.aureus suspension for 1 day. The infected wounds were treated with 50 μl of different samples carefully. The wound areas in the NIR group required an additional 3 min of exposure with the 808 nm NIR laser (1.0 W/cm2). Subsequently, the wound areas were photographed and measured every 2 days. The formula below was used to determine the relative wound area:All animal experiments in our work were performed under the guideline approved by the Institutional Animal Care and Use Committee of Huaqiao University and following the Administration of Affairs Concerning Experimental Animals of China. Female ICR mice (average body weight: 30 g) were randomly allocated into six groups: (i) control, (ii) BH, (iii) ICG, (iv) BI NPs, (v) ICG + NIR and (vi) BI NPs + NIR (n = 3). To establish the wound infection model, a dorsal skin wound (d = 6 mm) was created in each mouse and subsequently infected with in vivo, the exudate of the infected wound was taken every 2 days. The bacterial suspension was diluted with PBS before being placed on LB agar plates for incubation . Then, the colonies were formed and photographed.For analyzing the antibacterial activity of BI NPs The mice were euthanized on the ninth day, and the wounded tissues were gathered and stored with paraformaldehyde (4%). Then, they were examined using hematoxylin and eosin (H&E) staining and Masson’s trichrome staining and an optical microscope was used to capture the histological images.To evaluate the cytocompatibility of BI NPs, the mouse fibroblast cells (L929) were treated separately with different concentrations of BI NPs dispersion for 24 h. After that, the cell viability of each group was measured using Cell Counting Kit-8 (CCK-8), and the cell survival rate was calculated following the formula:The hemocompatibility was measured by fresh mouse blood. One milliliter of blood was centrifuged  and washed with PBS. Then, the erythrocyte suspension was diluted to 5% (v/v) and incubated with different concentrations of BI NPs dispersion at 37°C for 2 h. Next, the mixture was centrifuged to collect the supernatant, and its absorbance at 540 nm was determined. The hemolysis was determined following the formula:in vivo, the major organs of mice were stained with H&E staining and photographed with an optical microscope.To assess the biocompatibility of different samples BI NPs were obtained by self-assembling BH and ICG in an aqueous solution. The original morphology of unprocessed BH and ICG were irregular block crystals see . The masSEM and TEM were used to characterize the microstructure of BI NPs , BI NPs −1, the aromatic alkyl group) and ICG  in the FT-IR spectrum of BI NPs  of BI NPs dispersion and ICG solution were 66.4°C and 48.9°C, respectively. Notably, under the same irradiation conditions, the Tmax of BI NPs dispersion was higher than ICG solution, which could be due to the addition of BH showing no influence on the photothermal effect of ICG. Furthermore, the stable wrapping of ICG in the self-assembled nanoparticles also reduced its interaction with free radicals/ions in the solvent. max increased from 36.3°C (0.5 W/cm2) to 44.5°C (0.75 W/cm2) and 54.0°C (1.0 W/cm2) as the laser power density increased. The results showed that the increase in temperature was positively correlated with the laser power density. Different concentrations of BI NPs dispersion were irradiated with an NIR laser (0.75 W/cm2), and the photothermal conversion effects were recorded by an infrared imaging camera  group, 10.38% in the ICG (5 μg/ml) group, and 59.5% in the ICG + NIR group. Although different treatment groups inhibited bacterial growth in a concentration-dependent manner, the BI NPs + NIR group continued to display the highest inhibition rate among the other groups.Through the plate counting method, the eradication effect of BI NPs on ar plate . Each trE.coli  in response to the NIR laser. At the same time, BI NPs showed enhanced synergistic antibacterial activities in vitro compared to a single medication or PTT. Furthermore, in vivo, experiments demonstrated that BI NPs combined with NIR laser achieved long-term bacterial removal, accelerated wound healing, and reduced the risk of infection recurrence. In conclusion, this NIR-responsive nanoplatform was expected to provide a feasible approach for the repairment of infected wound.In summary, an NIR-responsive nanoplatform (BI NPs) was proposed for chemotherapy/PTT synergistic therapy and repairment of rbad076_Supplementary_DataClick here for additional data file.


Traumatic spinal cord injury (SCI) is a devastating neurological disorder often accompanied by neuropathic pain (NeP), significantly affecting patients' quality of life. This retrospective study aimed to investigate the impact of the time from injury to surgery on the development of NeP following traumatic SCI. Medical records of patients with traumatic SCI who underwent surgical intervention between January 2017 and January 2021 at two specialized centers were reviewed. Variables associated with NeP including demographics, injury profiles, medical history, surgical details, and pain assessments were investigated. Independent risk factors related to NeP were identified using multivariate logistic regression analysis. A total of 320 patients met the inclusion criteria, with 245 (76.6%) being male and a mean age of 56.5 ± 13.2 years. NeP was identified in 48.4% of patients (155 of 320). The multivariate analysis identifies age at injury, Injury Severity Score, and the neurological level of injury as independent risk factors for the development of NeP in both AIS A and AIS B, C, and D subgroups. Additionally, a significant association between the time from injury to surgery and NeP was observed in AIS B, C, and D patients, while no such association was found in AIS A patients. This study highlights the benefits of early and ultra-early surgical intervention in preventing NeP in patients with incomplete traumatic SCI , underscoring the importance of optimizing surgical timing to improve patient outcomes. Prospective studies are warranted to establish evidence-based surgical guidelines for managing traumatic SCI and preventing NeP effectively.The online version contains supplementary material available at 10.1186/s13018-023-04355-7. Traumatic spinal cord injury (SCI) poses a significant clinical and public health challenge, resulting in substantial morbidity and disability , 2. DespSevere neuropathic pain after SCI is recognized as one of the most distressing, debilitating, and incapacitating conditions, often leading to a lifelong and significant negative impact on a person's ability to participate in activities, recreation, and entertainment. This condition results in a compromised quality of life (QOL), long-term reliance on drug abuse, sleep disturbances, anxiety, depression, and reduced functionality , 12, 14.The proper time of surgical intervention of traumatic SCI has been an area of ongoing research. Surgeons have increasingly recognized the significance of secondary injury in SCI, leading to the growing recognition of the concept of “time is spine.” This concept supports the notion that early or urgent surgical intervention, including decompression and stabilization, can be safe and effective for traumatic SCI, restoring blood flow improving perfusion, and potentially mitigating the course of secondary injury , 15–17. Despite this progress in understanding the benefits of early surgical intervention for traumatic SCI, there remains a lack of data regarding its influence on the development of neuropathic pain after traumatic SCI. Hence, the current study aimed to address two objectives: (1) investigate the prevalence and status of NeP in patients with traumatic SCI, utilizing the Douleur Neuropathique 4 Questions (DN4) questionnaire from baseline to the final neurological follow-up and (2) determine the extent to which the time from injury to surgery affects the development of NeP after acute traumatic SCI. This research seeks to shed light on an important aspect of SCI management and contribute to better understanding and optimizing treatment strategies for individuals suffering from this condition.Consecutive adult patients (aged > 18 years) admitted to two specialized tertiary rehabilitation hospitals specifically catering to acute traumatic SCI  were retrospectively included between 2017.01 and 2021.01. Only patients who received surgical intervention within one week after sustaining the injury possessed complete medical records, had preoperative and postoperative pain assessments available, and underwent follow-up for a minimum of 12 months were considered eligible for inclusion. Exclusions were made for patients with non-traumatic spinal cord injuries (excluding single-event ischemia) and those who deceased during the primary rehabilitation period in the hospital. Furthermore, patients with peripheral nerve injury, diabetes mellitus, tumors, severe spasticity (Ashworth grade 3 or higher), or any other co-morbidity that could impact the neuraxial or peripheral nervous system were not included in the study. The study's protocols were duly approved by the relevant institutional research ethics board, and informed consent was waived.A comprehensive set of demographic data, encompassing age, gender, age at injury, education level, smoking, and alcohol habits, as well as the patient's past medical history and the Charlson Co-morbidity Index (CCI), were collected. Relevant SCI-related characteristics such as the injury level, time and mechanism of injury, time from injury to surgery, and Injury Severity Score (ISS) were meticulously collected. Regarding pain-related data, information on the severity and nature of pain, its specific location, factors that alleviate or exacerbate it, its impact on daily activities, frequency of accompanying symptoms, and treatments employed were diligently documented. Length of hospital stays (LOS) and hospitalization expenses were also recorded. For the analysis of time from injury to surgery, clinically relevant time intervals were chosen, with 8 and 24 h being the primary cut-offs. These specific timeframes have been frequently studied in patients with traumatic SCI .A neurological examination was performed following the International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI)  at a detFor the purposes of this study, SCI neuropathic pain (NeP) was defined as pain with a positive DN4 (4/10) rating, occurring at or below the level of injury and involving areas with sensory abnormalities as assessed by the DN4 questionnaire and the ISNCSCI examination. In this study, the Chinese version of the DN4 questionnaire  was indeAll medical records and questionnaires were systematically recorded and reviewed by two independent authors/observers, and any discrepancies were resolved through consensus or consultation with senior co-authors.Data were presented as mean ± standard deviation (SD) (or median with interquartile range (IQR), where applicable) for continuous variables and frequencies for categorical variables. The normality of distribution was tested by the Shapiro−Wilk test. In the complete cohort, using the Mann–Whitney U test for nonparametric variables, Student’s t test for parametric variables, and the χ2 test for categorical variables.p value < 0.1) in the univariate regression analysis or were deemed clinically relevant were included in the multivariate analysis. The Box-Tidwell test was applied to evaluate the assumption of linearity in the logit for continuous variables. Multicollinearity was assessed using the variance inflation factor in a multiple regression model with the same variables. The results were expressed as odds ratios (ORs) with their corresponding 95% CIs . The sample size in our study met the demands of events per variable during logistic regression analysis. A two-tailed p value < 0.05 was considered statistically significant. The statistical analyses were conducted using SPSS 26.0 , while GraphPad Prism (Version 8) was utilized for data visualization.Based on the significantly different neural recovery patterns and neurological outcomes between patients with complete injury (AIS A) and patients with incomplete injury  , 27, we n = 112), sensory incomplete , and motor incomplete  lesions. Neurological levels of injury were: high cervical (n = 101), low cervical (n = 136), thoracic (n = 36), and thoracolumbar (n = 47). The mean time from injury to surgery was 44.2 ± 29.7 h, 9.7% (31/320) of patients undergoing surgical intervention within 8 h from the time of injury, 20% (64/320) of patients undergoing surgical intervention within 24 h from the time of injury and the remaining 70.3% (225/320) having a surgical procedure after 24 h and up to 150 h after injury. Additional file From a total population of 363 patients who met our inclusion criteria, 35 were excluded due to incomplete data, and 8 were excluded due to insufficient follow-up, leaving 320 individuals with complete datasets in the final analysis Fig. . The meaP < 0.001; Table Out of 320 individuals, 155 (48.4%) presented with neuropathic pain  as their most severe pain, while at-level NeP could be identified in 60.0% (93/155) of these individuals. The timeline of NeP occurrence was also investigated, 32.9% (51/155) of the individuals developed NeP (DN4-positive) within 3 months post-injury and the remaining 67.1% (104/155) presented with possible neuropathic pain at the chronic stage (> 3 months). Mechanical allodynia was present in 22.6% of individuals, and thermal allodynia was present in 27.1% of individuals  and more severe neurological injury  than those without NeP. Patients with NeP recorded a significantly longer time from injury to surgery compared to patients without NeP (P = 0.023). It is worth noting that patients with NeP seem more likely to receive high-dose methylprednisolone therapy at the acute phase (p = 0.030). Differences were not observed in sex, BMI, etiology, and Neurological level of injury (p > 0.05).Patients with NeP were significantly older than those without NeP , ISS score (p = 0.020), neurological level of injury (p = 0.025) and treated with high-dose methylprednisolone , ISS score (p = 0.008), neurological level of injury (p = 0.038), treated with high-dose methylprednisolone (p = 0.007), and time from injury to surgery , and neurological level of injury (p = 0.013). NeP in AIS B, C, and D patients was consistently associated with a higher age at injury (p = 0.025), higher ISS score (p = 0.006), longer time from injury to surgery (p = 0.014), and history of high-dose methylprednisolone therapy (p = 0.002). Time from injury to surgery was not significantly associated with NeP in AIS A patients, however, AIS B, C, and D patients who had a surgical procedure performed within 8 h (ultra-early) of injury had significantly less NeP than those with surgery performed after 24 h (late) (p = 0.021), and patients who had a surgical procedure performed within 24 h (early) of injury had significantly less NeP than those with surgery performed after 24 h (late) (p = 0.039).Stepwise multivariate logistic regression analysis identified that NeP in AIS A patients was associated with a higher age at injury , delayed surgical intervention was identified as a key contributor to an augmented risk of NeP. These findings furnish compelling evidence reinforcing the advantages of early surgical intervention, notably ultra-early surgery, in mitigating the incidence of NeP after traumatic SCI.The reported incidence rate of NeP following traumatic SCI displays a wide range of 25–84% , 28, 29.Moreover, within the subset of patients experiencing NeP, a significant 60.0% were found to exhibit at-level NeP. Notably, and regarding the timeline of NeP occurrence, a substantial proportion (32.9%) of individuals developed NeP within 3 months post-injury, while the majority (67.1%) presented with possible neuropathic pain at the chronic stage (> 3 months). These findings underscore the critical importance of comprehending the temporal evolution of NeP in SCI patients, a factor that can significantly inform clinical management and intervention strategies. Furthermore, our analysis identified several factors consistently associated with the development of NeP in both AIS A and AIS B, C, and D subgroups. Specifically, older age at injury, higher Injury Severity Score (ISS), and more severe neurological injury were all found to be significantly linked to an elevated likelihood of NeP. These associations shed light on the substantial influence of injury severity and patient characteristics on the development of NeP.The principal focus of this study was to explore the potential impact of the time from injury to surgery on the occurrence of NeP in patients with SCI. Intriguingly, we observed that patients afflicted with NeP exhibited a significantly prolonged duration between injury and surgical intervention compared to those without NeP. This observation suggests a plausible association between delayed surgical intervention and the development of NeP. Earlier research has also reported a correlation between surgical timing and improved clinical outcomes in SCI patients , 31, 32.Additionally, our study has shed light on a compelling association, wherein patients experiencing Neuropathic Pain (NeP) were notably more prone to receiving high-dose methylprednisolone therapy during the acute phase. It is important to note that the employment of high-dose methylprednisolone is presently discouraged due to its significant systemic adverse effects . NeverthThis study represents the attempt at a comprehensive exploration of the factors underlying NeP in patients with traumatic SCI. The findings unveiled that delayed or late surgery may emerge as a noteworthy risk factor contributing to the presence of NeP. However, it is imperative to acknowledge the inherent limitations inherent in this study. Primarily, its retrospective nature introduces biases and constraints in data collection. To corroborate these findings, prospective studies with larger sample sizes and standardized protocols are indispensable. Secondarily, in the present study cohort, neuropathic pain was screened using the DN4 questionnaire as it was an ideal and validated tool for screening neuropathic pain , 42, theOur study proposes that incomplete AIS B, C, and D patients with acute traumatic SCI would benefit from surgical intervention performed within 8 and 24 h from time to injury, with a reduced risk of developing neuropathic pain compared to those who undergo surgical intervention later than 24 h. These findings provide evidence for the necessity of early surgical intervention, provide information for clinical decision-making, and strengthen the overall management of traumatic SCI. Efforts should be made to streamline the initial care of acute traumatic SCI patients to further reduce the time between injury and surgical intervention. Further research is warranted to elucidate the underlying mechanisms and potential therapeutic interventions to mitigate neuropathic pain in SCI patients.https://www.liebertpub.com/loi/neu [This study was not formally registered because this was a non-interventional study . The ana/loi/neu .Additional file 1: Table S1. Severity and neurological level of injury.


The differential diagnosis of benign and malignant VCFs is challenging. This study was designed to develop and validate a radiomics model to predict benign and malignant VCFs with p < 0.05) and test group , while there was no significant difference between the radiomics model and clinical–radiomics model (p > 0.05). The accuracies of the radiomics and clinical–radiomics models were 94.0% and 95.0% in the training group and 93.2% and 93.2% in the test group, respectively. The three models all showed good calibration . According to the decision curve analysis (DCA), the radiomics model and clinical–radiomics model exhibited higher overall net benefit than the clinical model.Twenty-six features (9 PET features and 17 CT features) and eight clinical variables  were ultimately selected. The area under the curve (AUCs) of the radiomics and clinical–radiomics models were significantly different from that of the clinical model in both the training group (0.986, 0.987 vs. 0.884, The PET/CT-based radiomics and clinical–radiomics models showed good performance in distinguishing between malignant and benign VCFs. The radiomics method may be valuable for treatment decision-making. Vertebral compression fractures (VCFs) are common clinical problems that can be caused by various reasons –4. AccorBoth open biopsy and percutaneous biopsy are invasive with complications of different degrees. Modern radiological imaging techniques, including computed tomography (CT), magnetic resonance imaging (MRI) and bone single photon emission computed tomography/computed tomography (SPECT/CT), may be helpful in the differential diagnosis based on morphology, signal intensity abnormalities, blood flow and metabolic status. MRI has been established as the most relevant imaging technique for the diagnosis of malignant vertebral lesions due to its high sensitivity to bone marrow abnormalities , 8. Some18F-fluorodeoxyglucose-positron emission tomography/computed tomography (18F-FDG-PET/CT) can provide information about both morphological changes and metabolic status of the diseased vertebra. In general, tumor-induced fractures accumulate 18F-FDG, while benign fractures are not expected to accumulate the same high level of 18F-FDG [ 18F-FDG , 10. Thi 18F-FDG . In addi 18F-FDG –16. Howe 18F-FDG , 18.To date, the diagnosis of the causes of VCFs by CT, MRI, bone SPECT/CT and PET/CT is still largely dependent on the experience of diagnostic physicians. Radiomics can extract a large number of quantitative features from digital medical images in high throughput and provide objective information that is difficult for human eyes to quantify –21. ManyThe objective of this study was to predict the benign and malignant nature of VCFs based on PET/CT radiomics and clinical indicators.Our institutional Ethics Review Board approved this retrospective study and waived the written informed consent requirement. The analysis was conducted on 439 patients diagnosed with vertebral compression fracture or pathological fracture after PET/CT in our hospital from January 2016 to January 2023, with a total of 539 vertebrae. Vertebral compression fracture was defined as (1) a reduction in vertebral height, (2) cortical discontinuity of an endplate or vertebral cortex with impaction into the vertebral body, or (3) buckling of the vertebral cortex , 23. Pat18F-FDG-PET/CT imaging was obtained by a GE Discovery710 instrument, USA. 18F-FDG was produced by the GE Minitrace cyclotron and FDG synthesis module, and the radiochemical purity was > 95%. All patients fasted for more than 6 h, and blood glucose was controlled below 11.1 mmol/L before injection. Patients received an intravenous injection of approximately 3.70–5.55 MBq/Kg body weight of 18F-FDG, and PET/CT scans were performed from the top of the skull to the upper femur (limbs were scanned if necessary) after a 60 min rest. The CT scanning voltage was 120 keV, tube current was 100 mAs, and the layer thickness was 3.75 mm. PET scanning was performed with three-dimensional acquisition, 2.5 min/ bed, and 5 ~ 7 beds were collected. An iterative method was used to reconstruct the image.Two nuclear medicine physicians with more than 5 years of experience, who were blinded to the lesion results, analyzed and recorded the lesion features, including lesion site, number, osteolytic destruction, fracture line, soft tissue mass/swelling, and appendices/posterior vertebrae involvement. If there was a disagreement, the decision was made by a third experienced nuclear medicine physician. Multiple metabolic parameters including SUVmax, SUVpeak, SULmax, and SULpeak were measured by creating a region of interest (ROI) along the lesion edge. Data on the patients’ age, sex and history of malignancy were also collected.https://www.slicer.org). Two experienced PET/CT diagnostic physicians manually drew the ROI and were blinded to the final diagnosis and clinical history of the patients. For each fractured vertebra, a three-dimensional ROI was drawn along the margin of the cortex of the whole vertebral body and the anterior margin of the bilateral pedicle on sagittal CT images of 3.75-mm thickness. The ROI of PET was sketched along the edges of the ROI of CT. A total of 107 features including 18 first-order features, 75 texture features and 14 shape features were extracted from the ROIs of PET and CT images using 3D Slicer’s built-in module SlicerRadiomics. There are five categories of texture features, including the gray-level dependence matrix (GLDM), gray-level co-occurrence matrix (GLCM), gray-level run length matrix (GLRLM), gray-level size zone matrix (GLSZM), and neighboring gray tone difference matrix (NGTDM). To ensure the stability and reproducibility of the acquired radiomics features, each set of PET and CT image data ROIs was segmented, and radiomics features were extracted twice. The intraclass correlation coefficient (ICC) for each radiomics feature was calculated. ICC > 0.75 was considered indicative of stability, and features were thereby entered into the statistical analysis that followed.The workflow of the radiomics analysis included lesion segmentation, radiomics feature extraction, feature selection, and model construction Fig. . PET/CT Student’s t test was conducted if the features were consistent with the normal distribution, and the Mann–WhitneyU test was conducted if the features were not. A value of p < 0.05 was considered statistically significant. For features with high repeatability, the Pearson’s standard correlation coefficient (defined as corr) was used to calculate the correlation among features. For those pairs of features showing high correlation coefficient (corr > 0.9), the feature with a higher p value was excluded and the maximum redundancy was deleted each time. Least absolute shrinkage and selection operator (LASSO) regression was applied with penalty parameter tuning by tenfold cross-validation, and features with nonzero coefficients were selected. Finally, the features of CT and PET after screening were entered into a support vector machine (SVM) classifier to establish a radiomics signature that distinguished between benign and malignant VCFs. A radiomics model was built based on both CT and PET findings.Student’s t test was used for normally distribution data, and the Mann–WhitneyU test was used for nonnormally distributed data. The Chi-square test was used for categorical variables. Statistically significant characteristics were selected. The same machine learning model in the clinical signature building process was used. Then, the clinical signature and radiomics signature were combined to develop a final clinical–radiomics prediction model. A clinical–radiomics nomogram was developed in this study.The building process of the clinical signature was almost the same as the radiomics signature. The performance of each model was assessed according to the area under the curve (AUC) based on the receiver operating characteristic (ROC) curve analysis, and the sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value(NPV) were calculated in the training and test groups. The Delong test was used to compare the AUC between the three models. Decision curve analysis (DCA) was used to determine the clinical benefit of each model.https://www.medai.icu), which is based on PyTorch 1.8.0. Continuous variables are expressed as the mean ± standard deviation(SD), and categorical variables are expressed as absolute counts and percentages(%). Statistical significance was defined as a two-sided p value < 0.05.Statistical analyses were performed using SPSS  and the “Onekey AI” platform (p < 0.05), but not in sex (p > 0.05).Among the 121 patients, 51 patients were diagnosed with benign VCFs, with 67 vertebral bodies , and 70 patients were diagnosed with malignant VCFs, with 77 vertebral bodies . The difference between benign and malignant VCFs was statistically significant in age , 6 cases were multiple myeloma, 5 cases were lymphoma, and 2 cases were Langerhans cell histiocytosis.Twenty-six features, consisting of 9 PET features (6 first-order features and 3 texture features) and 17 CT features  were selected to construct the radiomics model after LASSO regression and tenfold cross-validation  for the training group and 0.962  for the test group  for the training group and 0.858 for the test group  for the training group and 0.948  for the test group Fig. .p values of the Hosmer–Lemeshow test for the three models were 0.664, 0.787, and 0.422 in the training group and 0.241, 0.237, and 0.051 in the test group, respectively. The Delong test was used to compare the AUCs of the three models. In both the training and test groups, the radiomics model and clinical–radiomics model were significantly different from the clinical model (p < 0.05), but there was no significant difference between the radiomics model and clinical–radiomics model (p > 0.05). The DCA demonstrated that the radiomics model and clinical–radiomics model could provide higher overall net benefit than the clinical model , indicating that malignant VCFs were associated with higher metabolic activity on PET/CT.Conventional metabolic parameters of PET/CT such as max and peak of SUV and SUL have been used to quantify intratumoral heterogeneity . Some prAge was significantly different in distinguishing between benign and malignant VCFs. The mean age in the malignant VCF group was less than that in the benign VCF group, which was consistent with previous studies , 19, 24.p < 0.05) and the test group .Radiomics has been used in many clinical studies, including tumor molecular characteristics, patient prognosis and response to therapy . A numbeWe believe that radiomics-based models can improve the diagnostic accuracy and efficiency of diagnostic physicians because radiomics models use mathematical algorithms to describe lesions more objectively and can complement radiologists by providing quantitative information that is not available through visual analysis. Radiomics features are believed to reflect intraregional heterogeneity , 34. In This study has several limitations. First, this was a single-center retrospective study with a relatively small sample size, especially in the test group. Second, most of the malignant VCFs were metastatic tumors, while the proportion of myeloma and lymphoma was small, which might lead to potential confounding factors. Third, the slice thickness of 3.75 mm might be a limitation to this study. Thinner axial slices could increase reliability. Furthermore, we used radiomics for the analysis, and expanding the sample size for deep learning might make the results more reliable and meaningful. Finally, we only performed internal validation. Additional external validation is required to confirm the robustness and generalization of our model.18F-FDG-PET/CT can be used to predict the malignancy of vertebral compression fractures with high diagnostic accuracy. The predictive models can serve as potential decision support tools for clinicians and nuclear medicine physicians and help facilitate the appropriate management of patients with VCFs.In summary, the radiomics model and clinical–radiomics model combining clinical parameters with radiomics scores based on