The pathophysiology, diagnosis, and management of idiopathic normal pressure hydrocephalus (iNPH) remain unclear. Although some prognostic tests recommended in iNPH guidelines should have high sensitivity and high predictive value, there is often no positive clinical response to surgical treatment.In our study, 19 patients with clinical and neuroradiological signs of iNPH were selected for preoperative evaluation and possible further surgical treatment according to the guidelines. MR volumetry of the intracranial and spinal space was performed. Patients were exposed to prolonged external lumbar drainage in excess of 10 ml per hour during 3 days. Clinical response to lumbar drainage was assessed by a walk test and a mini-mental test.n = 12) (120.5 ± 14.9 ml) compared with the non-responder group .Twelve of 19 patients showed a positive clinical response and underwent a shunting procedure. Volumetric values of intracranial space content in responders and non-responders showed no statistically significant difference. Total CSF volume  was higher than previously published. No correlation was found between spinal canal length, CSF pressure, and CSF spinal volume. The results show that there is a significantly higher CSF volume in the spinal space in the responder group (This study demonstrates for the first time that CSF volume in the spinal space may have predictive value in the preoperative assessment of iNPH patients. The results suggest that patients with increased spinal CSF volume have decreased compliance. Additional prospective randomized clinical trials are needed to confirm our results. Normal-pressure hydrocephalus (NPH) is a form of chronic communicating hydrocephalus characterized by enlarged ventricles with normal or occasionally slightly elevated intracranial pressure. NPH can be of unknown cause  or secondary if known causes such as intracranial hemorrhage, trauma, central nervous system infections or intracranial neurosurgical procedures lead to its development , 2.iNPH is a chronic steadily progressing disease. It is the most common form of hydrocephalus in older adults and usually occurs in people over 60 years of age, equally in men and women , 3. Its In general, pathophysiological mechanism of hydrocephalus development is often not clear in both experimental animals and patients. This especially applies to particular forms of the disease such as unilateral communicating hydrocephalus and hydrocephalus conjoined with spinal pathology , 8, as wDespite its unclear nature iNPH is considered by many as a treatable cause of dementia and ataxia because a significant number of patients may improve clinically with ventricular shunting –19. ConvThe first step consists of being certain about correct diagnosis. However, despite current guidelines, the diagnostic criteria for iNPH are still unclear. Various invasive prognostic tests, such as tap test (large-volume lumbar puncture), prolonged external lumbar drainage with gait testing before and after CSF removal or CSF infusion testing for measurement of CSF outflow resistance are used to identify patients who are likely to respond to shunting surgery . It has Although some of the prognostic tests recommended in the iNPH guidelines should have high sensitivity and high predictive value, sometimes there is no positive clinical response to surgical treatment. Therefore, there are probably other factors that can significantly influence clinical outcome, one of them being the state of regional cerebral blood flow (CBF) before and after surgery. Recent studies in which non-invasive radiological techniques have been applied  clearly demostrated importance of regional CBF determination in preoperative iNPH patient selection , 22.Preoperative evaluation of iNPH patients usually involves lumbar drainage, which removes a certain volume of CSF from the CSF system without determining the overall starting CSF volume . Indeed, from the existing literature data, it can be concluded that the values of total CSF volume vary considerably . Thus, iThis study was conducted at the Department of Neurosurgery, General Hospital Varaždin, Croatia in the period from January 2017 to August 2019.The conducted study was voluntary and all data used during the study were previously protected, anonymized and stored in a database. The study protocol was approved by Ethics Committee of the Faculty of Medicine, University of Zagreb: October 30, 2017 , and Ethics Committee of the General Hospital Varaždin: January 20, 2017 (Number: 02/1-91/79-2017). The patients provided their written informed consent to participate in this study.Subjects were patients with clinical and neuroradiological signs of iNPH that came to our clinic for possible shunting procedure evaluation. Patient selection for neurosurgical treatment (ventriculoperitoneostomy) was performed according to the latest iNPH guidelines , 23. In Patients underwent MRI on GE 1.5T Signa HDXT  or Siemens Magnetom Aera 1.5 T  device. Brain MRI was performed in standard sequences and additional high-resolution sagittal T1 sequences of the head were taken for research purposes. The first MR device 1.5T GE Signa HDXT using FSPGR 3D (fast spoiled gradient echo) recorded T1 sequences  and the second MR device 1.5 T Siemens Magnetom Aera recorded T1-MPRAGE (magnetization prepared rapid gradient echo) sequences .Additionally, the first MR device recorded T2 3D Myelo sequences  of the spine, while the second MR device recorded 3D-T2 SPACE sequences  of the spine.High spatial resolution sequences were used, which provide a good contrast between the CSF and the surrounding tissue, i.e., enable clear detection of the edges of the cerebrospinal fluid space. MR imaging of the spine was performed in two segments due to its length: the upper segment includes the craniocervical junction, cervical and thoracic spine up to the T8-T9 level, while the lower segment includes thoracic spine from the T7 level to the sacrum. With this method of recording, the entire neural axis was covered and the resulting overlapping regions in the craniocervical and thoracolumbar areas were excluded from the volume calculation by computer processing of the images.http://www.microdicom.com), conversion to NIfTI format was performed with the dcm2nii program tool, which is part of the free MRICRON program package (https://www.nitrc.org/projects/mricron). The images were then compressed in zip format and uploaded to the volBrain platform. The volBrain system generates a data report in PDF format containing information about the volume of the intracranial cavity (ICC) and its parts , and information about some macroscopic areas such as cerebral hemispheres, cerebellum, and brainstem by processing anonymized data in NIfTI format. After automatic segmentation of the subcortical structures, their volumes are determined . Before data processing, the patient's DICOM images were anonymized using the MicroDicom Viewer program, and then converted to the NIfTI file format. After automatic contrast adjustment, the region of interest (ROI) is defined, and in the pre-segmentation procedure the edges of the spinal canal and spinal cord are detected using the threshold mode.Segmentation of the spinal space was performed using the program ITK-SNAP v3.8.0  to the end of the dural sac in the area of the sacral spinal canal.In patients with clinical and neuroradiological signs of iNPH, preoperative testing by CSF drainage was performed. In practice, usually two methods of testing are performed: lumbar puncture with large volume discharge (TAP test or large volume lumbar puncture—LVLP) or prolonged CSF drainage via lumbar drain. In this study, CSF pressure in lateral recumbent position before and after extraction of 15 ml of CSF was measured.The lumbar drain was inserted, lumbar drainage was opened and continuous release of the CSF was set at a rate of 10 ml/h (about 250 ml/day) for the next 3 days. During the entire test period, the patient's clinical condition was monitored by measuring cognitive and walking scores .t-test for related samples. The parametric Pearson and non-parametric Spearman methods were used to examine the relationship between the two variables. Differences confirmed at the level of p < 0.05 were considered statistically significant.Descriptive statistical analysis was used to analyze the values of lumbar CSF pressure and CSF volumes obtained by automatic and semi-automatic segmentation of the head and spine MR images. Values of the measured variables are presented in contingency tables, while the values of continuous variables are presented in the corresponding parameters—arithmetic mean and standard deviation. Comparation of the arithmetic means was examined by the In 19 patients with iNPH, MRI of the brain and entire spine was performed, followed by volumetric analysis of the cranial and spinal space. After a positive clinical response to prolonged external lumbar drainage , 12 patients underwent surgery (responders). There was no statistical difference in age between the responder  and non-responder group .n = 17). A comparison of the volumetric values of the intracranial space contents in responders and non-responders shows that there is no statistically significant difference between the two groups (p < 0.05).Anatomical parameters of the spinal space  were analyzed from MR images .The volume of the spinal CSF in all patients was 114.1 ± 21.4 ml on average, and the length of the spinal space was 59.1 ± 3.5 cm. Statistical analysis of the data indicates that in our sample there is no correlation between the volume of CSF in the spinal space and the length of the spinal space.p < 0.05) than in the non-responder group (103.1 ± 27.4 ml).2O, and from 254.8 to 594.1 ml, respectively.The correlation between the lumbar CSF pressure measured in the lateral recumbent position and the CSF volumes was examined. The data show  that CSFp < 0.05).A comparison of the intracranial, spinal or total CSF volumes with the level of lumbar CSF pressure measured in lateral recumbent position shows that there is no statistically significant correlation between lumbar CSF pressures and CSF volumes (p < 0.05) of the lumbar CSF pressure from 17.1 ± 3.7 cm H2O to 12.0 ± 3.1 cm H2O  (2O) and non-responder group (12.3 ± 3.4 cm H2O).After removal of 15 ml of CSF from lumbar subarachnoid space by lumbar puncture, there was a statistically significant drop ( n = 19) . There iIn this study of 19 patients with iNPH, we performed MR volumetry of the entire craniospinal space during the diagnostic preoperative procedure. The results of this study show that the group of patients who responded positively clinically to prolonged external lumbar drainage did not differ from the patients who showed no improvement in terms of standard radiological parameters measured  within the cranial space  and CSF It was previously believed that the total CSF volumes in infants vary from 40 and 60 ml, in older children between 60 and 100 ml, while it is approximately 150 ml in adults –29. The Analysis of the CSF volume data obtained in our study shows that the total CSF volume is much higher than previously published. CSF volume in the cranium varied between 111.4 and 447.4 ml (mean 305.1 ± 103.9 ml) , 2, wher2O; Compliance (C) is the ratio between the change in volume (ΔV) and the change in pressure (ΔP) and is usually described by an equation: C = ΔV/ΔP. Thus, in a highly compliant system, a large increase in volume results in only a small change in pressure, while in a less compliant system, a small increase in volume can result in a large increase in pressure. The compliant brain can accommodate a substantial increase in ventricular volume with a small increase in intracranial pressure. In clinical studies, compliance of the entire craniospinal space is usually determined, because compliance of the cranial and spinal spaces could only be determined separately if the cranial space were experimentally separated from the spinal space . In our Withdrawal of 15 ml CSF during lumbar puncture resulted in a decrease in CSF pressure in our patients , althougMany studies reveal that spinal space is crucial as a compensatory space for the regulation of CSF pressure and subsequently, brain perfusion. We have recently shown experimentally that the addition or extraction of a volume of fluid significantly alters intracranial CSF pressure in both horizontal and upright positions without significantly altering cranial CSF volume . A similAccording to all of the afore mentioned, it is likely that in our study, after lumbar puncture and an acute decrease in CSF volume of 15 ml, there was primarily a spinal decrease in CSF volume , associated with a decrease in CSF pressure throughout the CSF system, and an improvement in cerebral perfusion. Thus, we would expect that LP shunts reduce the spinal CSF volume, while VP shunts keep the cranial and spinal CSF volume in the physiological range. This would be in accordance with the clinical findings in patients with spinal CSF leak, who do not tolerate the upright position well, and in those with cranial CSF leak, who do not tolerate the horizontal position well. The detailed explanation of this effect was published in our previous papers , 38.This would also be consistent with studies using MRI to examine cerebral perfusion before and after the tap-test , 44. In Increased spinal CSF volume would selectively mean that the main compensatory compartment is almost exhausted. Our results suggest that patients with an average greater CSF volume responded well clinically to lumbar drainage. It appears that lumbar drainage in their case increased compliance, i.e., the compensatory capacity of the entire craniospinal space, and regulated CSF pressure fluctuations, thereby improving brain perfusion. Previously, it was observed that the decrease in ICP wave amplitude (indirect indicator of compensatory capacity) during external lumbar drainage was a powerful indicator . In patiAccording to the classical concept, there is constant CSF secretion, unidirectional circulation, and predominantly passive absorption through arachnoid granulations into the dural brain sinuses , 50, 51.The results of our study in iNPH patients suggest that the infusion test would probably indicate abnormal absorption in patients with large spinal CSF volume, as the infusion would cause a significant increase in pressure due to decreased compensatory capacity.The classical concept that iNPH is simply a form of CSF circulation disorder, in which there is an imbalance between CSF production and reabsorption, is probably not valid , 8, 58. The pathophysiology of iNPH is associated with impaired distribution of metabolites in the interstitium of the brain and, in general, with impaired blood circulation. Thus, it appears that in iNPH, impaired glymphatic circulation may lead to brain tissue damage and eventually dementia . It appePeriventricular white matter perfusion decreases with age due to various pathological processes such as atherosclerosis, hypertensive arteriolar sclerosis, and diabetes, which may decrease the efficiency of small-caliber perforating arteries and lead to deep white matter ischemia. Clinically apparent NPH could also be triggered by lacunar periventricular white matter infarctions due to abnormal CSF pulse dynamics , 65. NumCraniospinal space analysis based on iNPH patient MR images strongly suggests significantly larger values of CSF volume both inside the cranium and inside the spinal compartment from those available in the literature. No connection between the length of the spinal canal, the CSF pressure value and the measured spinal CSF volume has been detected. Larger mean spinal CSF volume is associated with positive clinical response to external lumbar drainage. It seems that patients with enlarged spinal CSF volume have decreased compliance as spinal space represents the key compensatory capacity of the entire craniospinal space.There are several limitations of this study. Our sample size was adequate for detecting the possible connection between average spinal CSF volume and a positive clinical response to extended external spinal CSF drainage. However, it should be noted that our predictive analysis asked for studies involving serial assessments in larger samples. Our study also did not include information regarding genetic factors  nor comorbid medical conditions or medications patients were taking, which may have influenced the results. The larger sample size and additional data regarding their comorbidities would also enable us to investigate the possible correlation between spinal CSF volumes of patients with predominantly cognitive deficit and those with predominantly motor disturbance. In our future studies we aim to extend our investigation in that direction.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving humans were approved by Ethics Committee of the Faculty of Medicine, University of Zagreb, Croatia Ethics Committee of the General Hospital Varaždin, Croatia. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.NK and MK designed and conceptualized the study. NK and IK conducted CSF pressure measurements and VP shunt operations. NK and MR performed MRI volumetry and calculated CSF volumes. MK, DO, NK, and IJ drafted the initial version of the manuscript and explained the presented results. All authors have read and approved the final draft of the manuscript.


To compare the biomechanical performance of the hybrid lumbar fixation technique with the traditional and cortical bone trajectory techniques using the finite element method.Four adult wet lumbar spine specimens were provided by the Department of Anatomy and Research of Xinjiang Medical University, and four L1–S1 lumbar spine with transforaminal lumbar interbody fusion (TLIF) models at L4–L5 segment and four different fixation techniques were established: bilateral traditional trajectory screw fixation (TT–TT), bilateral cortical bone trajectory screw fixation (CBT–CBT), hybrid CBT–TT (CBT screws at L4 and TT screws at L5) and TT–CBT (TT screws at L4 and CBT screws at L5). The range of motion (ROM) of the L4–L5 segment, von Mises stress of cage, internal fixation, and rod were compared in flexion, extension, left and right bending, and left and right rotation.P > 0.05). The peak stress of the internal fixation system in the TT–CBT group was significantly lower than the other three fixation methods in all five conditions except for extension, with a statistically significant difference between the CBT–TT and TT–CBT groups in the left rotation condition (P = 0.017). In addition, compared with the CBT–CBT group, the peak stress of the rod in the CBT–TT group decreased by 34.8%, 32.1%, 28.2%, 29.3%, and 43.0% under the six working conditions of flexion, extension, left bending, left rotation, and right rotation, respectively, but not statistically significant (P > 0.05).Compared with the TT–TT group, the TT–CBT group exhibited lower ROM of L4–L5 segment, especially in left-sided bending; the CBT–TT group had the lowest ROM of L4–L5 segment in flexion and extension among the four fixation methods. Compared with the CBT–CBT group, the peak cage stress in the TT–CBT group was reduced by 9.9%, 18.1%, 21.5%, 23.3%, and 26.1% in flexion, left bending, right bending, left rotation, and right rotation conditions, respectively, but not statistically significant (Compared with the TT–TT and CBT–CBT fixation methods in TLIF, the hybrid lumbar fixation CBT–TT and TT–CBT techniques increase the biomechanical stability of the internal fixation structure of the lumbar fusion segment to a certain extent and provide a corresponding theoretical basis for further development in the clinic. In contrast to cancellous bone, the cortical bone does not exhibit significant deformation and degeneration with increasing age . The corOther studies have reported the use of a hybrid fixation technique in transforaminal lumbar interbody fusion.  This technique allowed for the shortening of the fixation level and achieved spinal stability without invading the adjacent segment . HoweverConstruction and validation of the FE models were completed in the previous study . The matThe TLIF procedure was performed in Mimics 17.0  with decompression and fusion of the L4–5 vertebrae. The L4–L5 vertebral body was then internally fixed with the following combinations of internal screws: (1) TT–TT group, TT at both L4 and L5 segments .P < 0.05.Statistical methods SPSS 27.0 software was used to analyze and process the data. The means of quantitative data were expressed as mean ± standard deviation. Paired t-test was used for the analysis of variance. All results were considered statistically significant at P = 0.031).The ROM of L4–L5 segment was shown in Fig. P = 0.029), and between the TT–TT and CBT–CBT groups in left rotation (P = 0.002) and right rotation conditions (P=0.046).The von Mises stress of the cage was displayed in Fig. P = 0.017).The von Mises stress of the internal fixation system was displayed in Fig. The von Mises stress of the rod was displayed in Fig. The pedicle screw fixation technique, which improves fixation strength and fusion rates, has become the gold standard for spinal fusion , 24. HowThe CBT technique is a superior alternative to traditional pedicle screws in clinical applications. Santoni et al.  performeFinite element analysis, being a dependable and replicable simulation calculation method, has gradually played an indispensable role in the study of spinal diseases and implant biomechanics. The use of 3D finite element method to create a digital model can more accurately simulate various complex working conditions of the spine and calculate the stress distribution under different loads. This approach can aid in exploring the damage mechanism and reduce the time and effort required for repeated mechanical tests. Matsukawa et al.  conducteIn comparison with previous studies, the finite element model developed in this study offered the following advantages: (1) the L4–L5 lumbar segments were commonly affected by lumbar degeneration and other disorders, whereas previous studies mostly focused on the L3–L4 lumbar segments. Therefore, the selection of L4–L5 lumbar segments in this study made the finite element model more clinically relevant to the common sites of lumbar degeneration and other disorders. (2) The finite element model developed in this study offered stronger model integrity, a higher level of detail, and more complex modeling compared to previous studies. It was a comprehensive lumbar spine model, which included all lumbar spine structures from L1 to S1. To enhance the accuracy and realism of the model, the mechanical properties of various ligaments, such as the anterior longitudinal ligament, posterior longitudinal ligament, ligamentum flavum, supraspinous ligament, interspinous ligament, intertransverse process ligament, joint capsule, and even articular cartilage, were defined individually. The complexity of modeling resulted in an exponential increase in computational workload.  The majority of prior research only characterized the properties of the bone and disk structures in the L3–L4 lumbar segments, without fully accounting for the comprehensive force distribution throughout the lumbar spine and the intricate anatomical structures of the ligaments and synovial joints encompassing the lumbar spine. These factors had a significant influence on the modeling and analysis outcomes. (3) Selecting a larger number of experimental samples resulted in a relatively more representative sample. Specifically, this study used four osteoporotic lumbar spine anatomical specimens for scanning and modeling, whereas previous studies often only selected a single sample –35. (4) TLIF has been widely used for the treatment of degenerative lumbar spine diseases due to its advantages such as less invasive and good postoperative spinal stability. This procedure has shown good clinical application with complete decompression, short operative time, low bleeding, little damage to the spine, quick postoperative recovery, low complication rate, and preservation of most of the posterior vertebral column structures , 40. HowThe mobility of the fused segment often served as an indicator of the stability of the internal fixation system, and the finite element analysis conducted by Wu et al.  demonstrPrevious biomechanical studies , 42, 43 In the present study, the CBT–TT group exhibited the lowest range of motion (ROM) of the L4–L5 segment in flexion and extension compared to the other three groups. However, the ROM of L4–L5 segment was greater in both lateral bending and rotation, indicating that the ROM of L4–L5 segment in the CBT–TT group was superior in flexion and extension, but inferior in lateral bending and rotation. Compared to the TT–TT group, the TT–CBT group exhibited a reduced ROM of L4–L5 segment, particularly in left lateral bending ; however, there was no statistically significant difference when compared to the CBT–CBT group. These findings were consistent with previous studies on the biomechanical properties of the hybrid fixation technique using finite element analysis , 37, whiThe conventional TLIF procedure involves segmental decompression arthrodesis, interbody fusion implantation, and posterior pedicle screw fixation . The intWu et al.  used finMcLachlin et al.  found thNewcomb et al.  suggesteAs research on the pedicle screw–rod fixation method continues to expand, fragmentation of the von Mises stresses on the screw–rod contact surface has been documented in the literature. Song et al.  illustraThis study, however, still had certain limitations that were inherent to finite element analysis. First, the model established in this study was based on the scan data of human specimens. Compared with normal human bones, the scan data of cadaveric specimens were affected by the preservation conditions of specimens and sampling methods, which could potentially affect the analysis results. Secondly, despite the fact that this model had been modeled and processed as meticulously as possible, the impact of soft tissues such as muscles had been disregarded. Thirdly, this study simulated six kinds of motion states under different working conditions, while there were more complex compound motions and corresponding compound force states that had not been taken into account. Fourthly, the sample numbers were still relatively small, with variations in age, bone condition, and other factors among the specimens.In this study, the biomechanical characteristics of four fixation techniques, TT–TT, CBT–CBT, CBT–TT, and TT–CBT, were compared in transforaminal lumbar interbody fusion by using a three-dimensional finite element analysis modeling method. The aim of this research was to simulate the complete lumbar vertebrae and accessory structures as accurately as possible, in order to produce more realistic and credible data. Compared to TT–TT and CBT–CBT fixation methods, the hybrid CBT–TT and TT–CBT lumbar fixation techniques increased the biomechanical stability of the fixed structures within the lumbar fusion segment. This study took into consideration the pivotal factors pertaining to the biomechanical performance. However, further research data was required to provide additional support for the clinical application of the hybrid lumbar fixation technique.


The goal of the research was to study the levels of adipokines and their associations with unstable atherosclerotic plaques in patients with coronary atherosclerosis and abdominal obesity (AO). Methods: The study included 145 men aged 38–79 with atherosclerosis of the coronary arteries (CA) and stable angina pectoris II-III FC who were hospitalized for coronary bypass surgery (2011–2022). The final analysis included 116 patients. Notably, 70 men had stable plaques in the CA (of which 44.3% had AO), and 46 men had unstable plaques in the CA (of which 43.5% had AO). Adipocytokine levels were determined using multiplex analysis (Human Metabolic Hormone V3 panel). Results: In the subgroup of patients with unstable plaques, patients with AO had a GLP-1 level that was 1.5 times higher and a lipocalin-2 level that was 2.1 times lower, respectively. GLP-1 is direct, and lipocalin-2 is inversely associated with AO in patients with unstable plaques. Among patients with AO, the level of lipocalin-2 in patients with unstable plaques was 2.2 times lower than in patients with stable plaques in the CA. The level of lipocalin-2 was inversely associated with the presence of unstable atherosclerotic plaques in the CA. Conclusion: GLP-1 is directly associated with AO in patients with unstable atherosclerotic plaques. Lipocalin-2 is inversely associated with unstable atherosclerotic plaques in patients with AO. Obesity, as an independent disease or a syndrome, and related metabolic disorders are an urgent problem of modern medicine since they lead to several serious diseases development, including atherosclerosis and cardiovascular diseases (CVD) . AbdominAdipokines are able to support chronic inflammation . ChronicMany studies combined in a large review mention the relationship between glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) and inflammation, emphasizing the importance of GIP and GLP-1-dependent pathways in atherosclerosis and coronary heart disease, and say that GIP and GLP-1 can be used as markers of the frequency, clinical course and recurrence of coronary heart disease and are associated with the degree and severity of atherosclerosis and myocardial ischemia . NumerouThe presence of dyslipidemia in patients of both groups was taken into account when the level of lipids and lipoproteins increased above the optimal value . Based op = 0.044) and a lipocalin-2 level that was 2.1  times lower, respectively.At the next stage, all the studied parameters were included in the model of logistic regression analysis of AO and adipocytokine associations in patients with unstable plaques . AO was According to the results of logistic regression analysis, GLP-1 is directly and lipocalin-2 is inversely associated with the presence of AO in patients with unstable atherosclerotic plaques. All the studied indicators were included in the ROC analysis with an assessment of sensitivity and specificity.p = 0.044).Given the poor quality of the model for lipocalin-2 (AUC 0.306), the results of the ROC analysis can be concluded only for GLP-1 . The levp = 0.008) than patients with stable plaques in CA.In patients with AO, patients with unstable plaques had a level of lipocalin-2 2.2 times lower , depending on the level of lipocalin-2, in which the level of lipocalin-2 was inversely associated with the presence of unstable atherosclerotic plaques in the coronary arteries .During the ROC analysis to determine the threshold value of lipocalin-2 for the presence of atherosclerotic plaque instability, an unsatisfactory quality model (AUC 0.306) was obtained.Glucagon-like peptide-1 (GLP-1) is the main incretin hormone that is secreted from the L-cells of the small intestine and is derived from the transcription product of the proglucagon gene . GlucagoFasting and post-meal GLP-1 levels were studied in adults and children with diabetes, obesity, and cardiometabolic risk factors. Previously, some cross-sectional studies reported that fasting GLP-1 levels were higher in adults with DM2 and obesity, while others found no difference. A recent study reported a significant increase in fasting GLP-1 levels in obese adult women ,23. It iOur results can also be explained by the phenomenon of GLP-1 resistance, which is caused by excessive visceral fat . GrassetData from the study by Albinsson-Stenholm E. et al. contradict the lack of GLP-1 release as a mediator of increased glucose levels in early manifestations of insulin resistance in white people. They found that fasting levels of GLP-1 correlated positively with serum insulin in the total cohort. However, this relationship was lost when data were corrected for age, sex, fasting glucose, and BMI. This confirms the complicated relationship between BMI and insulin when age and sex are added to the equations. They speculate that increased GLP-1 levels in subjects with hyperinsulinemia can be a compensatory mechanism in insulin resistance that occurs before glucose levels are highly elevated .In our study, in a subgroup of patients with unstable plaques, patients with AO had a GLP-1 level that was 1.5 times higher; GLP-1 is directly associated with the presence of AO in patients with unstable atherosclerotic plaques, while the level of GLP-1 ≥ 396,114 pg/mL with a sensitivity of 75% and a specificity of 68% is associated with the presence of AO in patients with unstable plaques in the CA. We understand that GLP-1 has well-known anti-atherosclerotic properties and that treatment with GLP-1 agonists reduces food intake, and thus these drugs are used in the treatment of obesity. However, in our sample of patients with coronary atherosclerosis, we received contradictory results, which may supplement the known information on this topic in white people. More in-depth research in this area is needed; it is possible to study the intestinal microbiota, and the genetic predisposition of GLP-1 resistance in patients with established coronary artery disease, especially those with unstable atherosclerotic plaques.Lipocalin-2 is a small lipid-binding protein with still unknown ligands , which i2, lipocalin-2 may also depend on fluctuations in creatinine and renal function, which requires a more thorough analysis. The study limitations also include a small sample size and the inclusion of only males.In this study, data were obtained that the levels of lipocalin-2 are 2.1 times lower in the subgroup of patients with unstable plaques and AO compared to patients with unstable plaques without AO. Lipocalin-2 is inversely associated with the presence of AO in patients with unstable atherosclerotic plaques. Among patients with AO, the level of lipocalin-2 in patients with unstable plaques was 2.2 times lower than in patients with stable plaques in the CA. If we look at the average values of lipocalin-2 in healthy people and people with coronary atherosclerosis, it can be noted that in our study the level of lipocalin-2 was several times higher, which is consistent with the generally accepted data on its pro-inflammatory properties. Thus, we obtained differences only in a subgroup of obese patients between stable and unstable plaques, which requires further study . This diThus, this study showed that GLP-1 is directly associated with the presence of AO in patients with unstable atherosclerotic plaques; the levels of lipocalin-2 are inversely associated with the presence of AO in patients with unstable atherosclerotic plaques. These results are contradictory but may bring out some issues that deserve further investigation in future research.The design of the study is a single-stage observational study. The study was conducted within the framework of joint scientific research of IIPM–Branch of IC&G SB RAS and FSBI “National Medical Research Center named after E. Meshalkin” of the Ministry of Health of the Russian Federation. The study was approved by the Local Ethics Committees of both institutions . The data and samples were collected after receiving written informed consent from all participants.The study included 145 men aged 38–79 years (mean age 62.28 ± 8.19) with coronary angiographically verified atherosclerosis of the coronary arteries, without acute coronary syndrome (ACS), with stable angina pectoris II-III FC, hospitalized in the clinic of the FSBI National Medical Research Center named after E. Meshalkin of the Ministry of Health of the Russian Federation for coronary bypass surgery (in the period from 2011 to 2022) .The inclusion criteria at the stage of preliminary selection of patients are as follows: male, diagnosis of coronary heart disease (CHD) verified by coronary angiography data, the presence of a history of myocardial infarction (MI) or episodes of stable angina pectoris documented by a description of the clinical picture of the disease, the results of ECG, and biochemical blood tests.2 (CKD-EPI)), respiratory failure, and liver failure), known active oncological diseases, and toxic lesion heavy metals.The non-inclusion criteria at the pre-selection stage of patients are as follows: female, the presence of ACS less than six months before admission (unstable angina or MI), clinically significant severe concomitant pathology in exacerbation  and standard hematoxylin-eosin and Van Gieson staining were studied on an Axiostar Plus binocular microscope. The study of fragments of intimate media revealed the presence of stable and unstable atherosclerotic plaques. The unstable plaque was differentiated according to the following criteria: the thickness of the fibrous covering is less than 65 microns, infiltration by macrophages and T-lymphocytes (more than 25 cells in the field of vision 0.3 mm), and a large lipid nucleus (more than 40%) .After the intervention, six patients dropped out of the study for the following reasons: voluntary refusal to participate, inability to contact the patient, and development of complications. In 23 patients, the type of plaques could not be determined using histological analysis. The final analysis included 116 patients. Of the studied group, 70 men had stable plaques in the coronary arteries (60.3%), and 46 men had unstable plaques in the coronary arteries (39.7%).Before the operation, biological material (blood) was taken. Biochemical studies were carried out in the Laboratory of Clinical Biochemical and Hormonal Studies of Therapeutic Diseases of IIPM–Branch of IC&G SB RAS. By multiplex analysis using the Human Metabolic Hormone V3 panel , adipocytokine levels were determined on a Luminex MAGPIX flow fluorimeter: Glucose-dependent insulinotropic polypeptide (GIP), Glucagon-like peptide-1 (GLP-1), Glucagon, Insulin, Leptin, Adiponectin, Adipsin, Lipocalin-2, and Resistin.2); Waist circumference (WC) was measured in a standing position, midway between the lower edge of the chest and the crest of the ilium along the middle axillary line. Abdominal obesity (AO) was established at a WC of more than 94 cm for the Caucasian race [The study took into account demographic characteristics, anamnesis of the disease, and the presence of chronic diseases . Patients underwent anthropometry, including height, body weight, waist, and hip circumference. Their body mass index was determined using the following formula: BMI = Body weight (kg)/Height  and standard deviation (SD), with nonparametric distribution as the median, 25, and 75 quartiles. A comparative study of variables with parametric distribution was carried out using the Student’s t-test. In the case of a nonparametric distribution, the Mann–Whitney U-test (for two independent groups) and the Kruskall–Wallis criterion were used. Spearman’s rank correlation coefficient (rs) was used to analyze the dependence of quantitative features of sample data from aggregates. In the case of nominal and ordinal data scales, cross-data tables and Pearson’s χ2 criterion were used and adjusted for probability . Associations were evaluated using multiple logistic regression analysis performed under the following conditions: the dependent variable is dichotomous; independence of observations; absence of multicollinearity, i.e., situations wherein independent variables strongly correlate with each other (r > 0.9); linear dependence between each independent variable and the logarithm of the odds ratio (logarithmic coefficients); and independence of residuals. The results of multiple logistic regression analysis were presented as OR and 95% CI for OR. To assess the threshold value of the diagnostic test, ROC analysis, sensitivity, and specificity assessment were carried out. The significance level was set at GLP-1 is directly associated with AO in patients with unstable atherosclerotic plaques. Lipocalin-2 is inversely associated with the presence of unstable atherosclerotic plaques in patients with AO. Further studies are needed to study lipocalin-2 and GLP-1 in patients with coronary atherosclerosis in this patient population.


The pathophysiology of lacunar infarction is an evolving and debated field, where relevant information comes from histopathology, old anatomical studies and animal models. Only in the last years, have neuroimaging techniques allowed a sufficient resolution to directly or indirectly assess the dynamic evolution of small vessel occlusion and to formulate hypotheses about the tissue status and the mechanisms of damage. The core–penumbra concept was extensively explored in large vessel occlusions (LVOs) both from the experimental and clinical point of view. Then, the perfusion thresholds on one side and the neuroimaging techniques studying the perfusion of brain tissue were focused and optimized for LVOs. The presence of a perfusion deficit in the territory of a single small perforating artery was negated for years until the recent proposal of the existence of a perfusion defect in a subgroup of lacunar infarcts by using magnetic resonance imaging (MRI). This last finding opens pathophysiological hypotheses and triggers a neurovascular multidisciplinary reasoning about how to image this perfusion deficit in the acute phase in particular. The aim of this review is to summarize the pathophysiological issues and the application of the core–penumbra hypothesis to lacunar stroke. The hypothesis underlying hyperacute stroke treatment both in the early and late time window is the presence of a vessel occlusion causing an hypoperfusion in the supplied territory for a variable time and with different degrees, ranging from salvageable tissue (penumbra) to irreversibly damaged tissue (core). The core–penumbra hypothesis was elaborated and proposed about 40 years ago . The proAdvanced neuroimaging techniques have been increasingly included in stroke management pathways and therefore practically used, relying on their estimation of the core–penumbra ratio or mismatch for selecting patients for treatment. Both computed tomography (CT)-based and magnetic resonance imaging (MRI)-based protocols have been used in several studies and in real practice, with CT-based advanced neuroimaging generally being more widely accessible than MR-based neuroimaging for the assessment of acute stroke. In general, advanced neuroimaging techniques use a sequentially angiographic approach  for assessing the presence of a large vessel occlusion (LVO) and/or a distal vessel occlusion (DVO) amenable for endovascular treatment, and a perfusion approach to evaluate tissue viability in the corresponding territory, particularly in the late time window. The quantification of core–penumbra mismatch is usually performed by using automated processing software with predefined perfusion thresholds . The preSince the first proposal of the penumbra concept, its application has been directed almost exclusively at the LVO model and this has resulted in the fact that both neuroimaging techniques and perfusion thresholds as well as the postprocessing software have been optimized for this setting. The occlusion of a small vessel, which is the primum movens of a lacunar stroke, has been substantially neglected by this model. For a long time, it was assumed that, since it is the occlusion of a terminal artery, without the possibility of compensation by collaterals, there is no pathophysiological possibility of a penumbra and its subsequent identification. This assumption is only partially true and has been redefined over the years, leading to the study of the perfusion status in lacunar stroke and to a better understanding of the pathophysiology of this stroke subtype. In this review, we deal, in a pragmatic way, with the perfusion aspects of lacunar stroke and their impact both on the knowledge of the physiopathology of this widespread and neglected subtype of stroke and on therapeutic choices in the acute phase.Before introduction of the penumbra concept, i.e., a dynamically progressive cerebral ischemia over an uncertain and variable period of time, the common thought was the immediate death of brain tissue after stroke onset. Lindsay Symon  was the The following step was the demonstration that increased oxygen extraction fraction marked potentially salvageable tissue using positron emission tomography (PET) ,10. StudComing back to a metabolic point of view, the brain accounts for only 2% of body mass, but it consumes approximately 20% of the body’s total basal oxygen consumption at rest. The high-energy need of the brain is the reason why a sufficient blood supply has to be maintained in all circumstances and it is supplied by 16% of the cardiac blood output. The mean number of neurons of a normal adult male’s brain is 130 billion (21.5 billion in the neocortex) . The braThe different energy requirements lead to different thresholds of energy consumption and, consequently, blood flow required for preservation of neuronal function and morphological integrity. Two critical levels of decreased perfusion were demonstrated in experimental studies: 1) a flow threshold for reversible failure of cellular functions ; (2) a lower threshold below which the membrane processes are irreversibly lost and morphological damage occurs as infarction  a flow t. The maiBased on the threshold concept of brain ischemia, the penumbra can be measured and localized using predefined flow thresholds on quantitative flow images. In experimental studies, this concept has been applied by using a direct imaging approach of threshold-dependent biochemical disturbances on brain slices. This approach allows for distinguishing the mismatch between processes that occur only in the infarct core and others that also affect the penumbra. The most reliable way to localize the infarct core is the loss of ATP on bioluminescent images of brain tissue. Tissue acidosis is a biochemical marker of core plus penumbra. The penumbra is the difference between the respective lesion areas. The inhibition of protein synthesis starts at a higher flow level .As time goes on and the brain remains hypoperfused, the infarct progresses through three phases  Table 1Table 1.In animal experiments, 3 h and 6–8 h after MCA occlusion, respectively, more than 50% and almost the entire penumbra became part of the irreversibly damaged core. However, some small regions characterized by reduced blood flow and preserved oxygen consumption could be observed for up to 24 h or even more around the lesion at the border zone of the ischemia ,30.Year by year, the development of treatment options for acute stroke led to cancel pathophysiological studies on the evolution of the core–penumbra relationship. Concurrently, metabolic imaging techniques have given way to morphological imaging, CT perfusion (CTP) and perfusion MRI. The concept of the penumbra in its practical application to the management of the hyperacute phase of stroke has also changed. Nowadays, the penumbra is generally considered in a dynamic approach and it associates time-sensitive hypoperfused brain tissue with decreased oxygen and glucose availability, still salvageable by intervention, restoring the blood flow; therefore, the penumbra is the potential target for neuroprotection in focal stroke . MoreoveThe revisited concept of penumbra fate  started All this information helps to define the concept of the penumbra at the tissue level and the information deriving from neuroimaging investigations substantially represent a surrogate of what happens at the cellular level or of the measurable metabolism in selective areas of the brain using PET techniques. However, the focus of the core–penumbra model is stroke due to LVO and the involvement of a large volume of brain tissue. The transferability of these concepts, including blood flow thresholds and timing, to different subtypes of stroke, particularly small vessel disease-related stroke, is not immediate.Lacunar stroke accounts for 25% of ischemic strokes . It is aA summary of the definitions is provided in Another source of variability comes from the etiological classifications of ischemic stroke, where a mixture of clinical, neuroimaging and histopathological issues are combined . The TriMoreover, there is a histopathological definition, and it has many potential pathophysiological implications, which are detailed in the next sections.The most used and convincing histopathological definition is provided by Poirier and Derouesné : (1) typC.M. Fisher was the first to note that most lacunes were distal to occlusive lesions of small perforating arteries ,64,65,66Lipohyalinosis is the major driver of SVD, and it is typical of lacunar stroke. Miller-Fisher proposed the definition of “hypertensive cerebral vasculopathy”, describing it as occlusion of the lumen of an artery, usually <200 mcm in diameter, whose wall structure is destroyed and highly fragmented, surrounded by hemosiderin-filled macrophages. The reorganization of a small perforating branch occlusion may include retrograde occlusion  and the formation of small “channels of recanalization”. Finally, a histopathological progression of lipohyalinosis encompasses three stages : (1) preThe histopathology provides relevant information but its main limitation for inferring the mechanisms of acute lacunar infarcts is due to the fact that autopsy studied old, affected vessels . ConversThe histopathology of brain tissue provides different and complementary information. The findings of Miller-Fisher’s examination are that chronic lacunes had trabeculated cavities surrounded by gliosis and few macrophages, while recent lacunes contained numerous macrophages and had minimal gliosis. The wide range of histopathological morphology solicited the proposal of a classification schema in 1984 : (1) typThe etiology of type Ib parenchymal lesions has been a matter of debate because it has implications for therapeutic targets. Some authors proposed that type Ib lesions are secondary to local edema instead of ischemia , describIn lacunar infarct, the process of injury (ischemia or infarct), repair and recovery are not static but highly dynamic. When the brain parenchyma loses its blood supply to a degree that affects its functioning but does not cause necrosis (as in the penumbra), an ischemia occurs. On the contrary, the parenchyma, when apoptosis and necrosis occur, is called an infarct. Ischemia and infarct represent the ends of a spectrum, balanced by continuous blood flow changes. There is increasing evidence supporting the role of decreased blood flow in lacunes, small subcortical infarcts and white matter hyperintensities (WMHs), as already known in large vessel occlusions, but with different features. A recent study showed decreased CBF and vascular reactivity in WMHs versus homologous contralateral normal-appearing white matter . The decFinally, the repair process may provide some useful information, but it is not easy to study it in vivo outside of animal models. Serial neuroimaging studies ,89,90 haIt is possible that a combination of involvement in lower-order branched vessels and progressive worsening of vessel wall disease/increasing number of involved vessels creates flow-limiting areas of vessel disease contributing to downstream hypoperfusion and WMH or lacune evolution .Small subcortical infarct is caused by the occlusion of a chronically and progressively thickened small perforating artery or by plaque in the parent artery ,91. AccoCollateral flow plays a relevant role in RSSI too. Indeed, if, in the past, lacunar infarction was thought to be caused by the occlusion of a terminal penetrating artery, without collateral circulation and the occurrence of penumbra ,101, on p = 0.002), confirming an inverse mismatch between hypoperfusion and infarct. On day 2, mean DWI lesion volume increased to 2.78 mL (SD 1.39 mL) and inverse mismatch was evident in all patients (p < 0.0001). Final lesion volume (FLV) at day 6 ranged from 0.64 to 6.0 mL  and it was a median 1.69 times larger than the initial DWI lesion and 3.63 times larger than the initial MTT lesion . Compared with final infarct volume, the initial DWI lesion was 55.7 FLV% (SD 34.3%) and the mean initial MTT deficit was 24.7 FLV% (SD 21.6%). Contrary to lesion growth using DWI, the MTT deficit showed no change (p = 0.98) from day 1 (24.7 FLV%) to day 2 (23.6 FLV%).It is possible that a perfusion evaluation distinguishing the penumbra from the core is not possible for RSSI in the same way it is performed for small and large territorial infarctions. The resolution of CTP maps does not allow for this task, as well described in Garcia-Esperon 2021 , where, Rudilosso et al.  providedIn general, although there were technical differences in the individual studies, time-based maps were found to be more sensitive than flow-based maps.The perfusion techniques in the hyperacute phase of stroke were studied and their scope of application and thresholds aimed at identifying patients amenable to IVT and EVT treatment, overcoming the concept of time and starting from physiopathology of the ischemic damage of the brain tissue involved. As previously said, the premise of these studies is the presence of a territorial ischemia conditioned by an LVO or DVO. Lacunar stroke, being caused by the occlusion of a single small perforating artery or by atherothrombotic involvement of the corresponding parent artery, is outside this scope, so there is no reliable information on the use of perfusion techniques in the hyperacute phase for the identification of a mismatch in the territory of a perforating artery.Indeed, lacunar perfusion lesions tend not to be visible on postprocessed core–penumbra maps, mainly because core–penumbra maps are typically smoothed on automated software including only relatively large groups of hypoperfused pixels in the core–penumbra lesion. Previous small studies suggested that visual assessment of the MTT map had reasonable sensitivity for detecting lacunar infarction ,113,114.p = 0.007), the largest initial and final infarct volumes (p < 0.001 and p = 0.029), the lowest relative CBF , and the lowest rate of anterograde and retrograde collaterals . Anterograde collaterals were associated with higher relative CBV  and a higher rate of deep cerebral microbleeds , whereas retrograde collaterals were associated with higher systolic and diastolic blood pressure (p = 0.031 and 0.020), smaller initial infarct volume  and a higher rate of lobar cerebral microbleeds . These findings suggest that anterograde collaterals play a main role in maintaining adequate microvascular perfusion after RSSI. Anterograde collaterals may come directly from partially occluded arteries or indirectly from anastomosis of the proximal segments of penetrating arteries, but the first case is more likely because of the fewer proximal anastomoses from basilar arteries and MCA documented in anatomical studies [Moreover, small perfusion defects seen in CTP and perfusion MRI, indicating the existence of penumbra, were associated with the development of early neurological deterioration (END) in a few series ,110,118. studies . The ade studies ,121.The impact of the identification of a perfusion deficit in CTP in the acute phase of stroke is one of the treatment decisions, in particular IVT. Conversely, in the late window, the lack of identification of a perfusion deficit usually does not allow for the performance of IVT. As detailed in the systematic review on CTP in lacunar stroke patients  and in tThe pathophysiology of lacunar stroke is complex and involves collaterals and perfusion status with penumbra at a smaller scale than stroke due to LVO. Hemodynamic factors play a role in the occurrence and evolution of RSSI and WMH and the exact mechanisms are not yet fully understood. Better knowledge of these issues would allow these processes to be targeted for focused therapeutic strategies.


However, whether Aβ42 and amyR have different meanings and whether Aβ40 represents more than an Aβ42-corrective factor remain to be clarified. Our study aimed to compare the ability of Aβ42 and amyR to detect AD pathology in terms of p-tau/Aβ42 ratio and brain glucose metabolic patterns using fluorodeoxyglucose-positron emission tomography (FDG-PET).Despite the high sensitivity of cerebrospinal fluid (CSF) amyloid beta (Aβ)42 and normal p-tau (A + T −  = 98) or pathological p-tau levels (A + T +  = 65) and 36 control subjects (A − T −). A + T − patients were further stratified into those with normal (CSFAβ42 + /amyR −  = 46) and pathological amyR (CSFAβ42 + /amyR +  = 52). We used two distinct cut-offs to determine pathological values of p-tau/Aβ42: (1) ≥ 0.086 and (2) ≥ 0.122. FDG-PET patterns were evaluated in a subsample of patients (n = 46) and compared to 24 controls.CSF biomarkers were analyzed with EUROIMMUN ELISA. We included 163 patients showing pathological CSF Aβ40 levels were the lowest in A − T − and in CSFAβ42 + /amyR − , higher in CSFAβ42 + /amyR + and highest in A + T + , resembling CSF levels of p-tau . We found a positive association between Aβ40 and p-tau in A − T − , CSFAβ42 + /amyR − , and CSFAβ42 + /amyR + patients  but not in A + T + . Investigating biomarker changes as a function of amyR, we observed a weak variation in CSF p-tau (+ 2 z-scores) and Aβ40 (+ 0.8 z-scores) in the normal amyR range, becoming steeper over the pathological threshold of amyR . CSFAβ42 + /amyR + patients showed a significantly higher probability of having pathological p-tau/Aβ42 than CSFAβ42 + /amyR − , which however still showed pathological values of p-tau/Aβ42 in some cases  unlike A − T − . Accordingly, we found reduced FDG metabolism in the temporoparietal regions of CSFAβ42 + /amyR − compared to controls, and further reduction in frontal areas in CSFAβ42 + /amyR + , like in A + T + .CSF Aβ42 and FDG hypometabolism typical of AD can be found in patients with decreased CSF Aβ42 levels alone. AmyR positivity, associated with higher Aβ40 levels, is accompanied by higher CSF p-tau and widespread FDG hypometabolism.Pathological p-tau/AβThe online version contains supplementary material available at 10.1186/s13195-023-01291-w. Holding scans with both typical and possible patterns as AD-positive, Cohen’s k analysis demonstrated a significant agreement between abnormal FDG-PET and pathological CSF p-tau/Aβ42 (Cohen’s k = 0.73), with a mismatch of 6% between them.Demographics of the participants to the substudy are reported in Additional file 42 + /amyR − patients showed a significant reduction in brain glucose consumption in a wide cluster encompassing the left parietal (BAs 19 and 40), temporal (BA 37), and frontal lobes   .Comparisons between FDG-uptake patterns of each patient group versus CG showed a significant reduction in the temporo-parietal and frontal regions  associated with a parallel increase in tau phosphorylation. Conversely, in our A + T + group tau pathology seems to proceed independently from amyloid pathology as no association was found between amyloid and tau CSF biomarkers. In line with our results, it has been recently demonstrated that the Aβ-induced increase in CSF p-tau levels might play a key role in initiating tau aggregation and spreading in early AD, while local tau seeding and auto-replication predominate once soluble p-tau concentrations reach a plateau in AD dementia [The specific meaning of the altered CSF Aβnditions . In our dementia .42 + and amyR + hold different meanings in AD pathophysiology, we considered patterns of brain glucose metabolism. Indeed, specific FDG-PET hypometabolic patterns have been demonstrated to predict AD dementia [42 + /amyR − and CSFAβ42 + /amyR + .To support our hypothesis that CSFAβdementia –17 and m42 + /amyR − patients and controls, we found a pattern of cortical hypometabolism encompassing the parietal and frontotemporal regions. Thus, the decrease in CSF Aβ42 alone may be associated with synaptic dysfunction and reduced brain glucose uptake in areas typically involved in AD [First, when comparing FDG-PET scans from CSFAβed in AD , 46. Howed in AD –49.42 + /amyR + showed further involvement of the frontal regions, configuring an FDG-PET pattern indistinguishable from A + T + . Previous studies have shown that soluble Aβ40, but not Aβ42, extracted from the frontal cortex of patients with AD gradually increases along with the progression of Braak scores, suggesting a specific link between Aβ40 levels and the progression of tau pathology [42 and increased Aβ40 levels, is associated with widespread brain hypometabolism, and probably with the spread of tau pathology, even in the absence of elevated tau proteins in the CSF [In contrast, patients with CSFAβathology . Moreoveathology . Our fin the CSF .181 in our cohort does not exclude a possible increase of other p-tau isoforms . Moreover, a threshold-based evaluation of CSF p-tau, as for other fluid biomarkers, can have limitations tied to the risk of false negative results, whose occurrence also encompasses pre-analytical  and analytical variables  [The lack of detection of elevated levels of CSF p-tauitivity) . On the 42 and low Aβ40 (CSFAβ42 + /amyR −) that differ from healthy controls for showing typical AD patterns of cerebral hypometabolism, and a second group with reduced Aβ42 and high Aβ40 (CSFAβ42 + /amyR +) showing widespread reduction of brain glucose uptake, undistinguishable from biologically defined AD patients (A + T +).Our results show that among patients classified as A + T − , there is a group with reduced Aβ42 + /amyR − and CSFAβ42 + /amyR + patients, as well as the first to examine differences and overlap between them, healthy controls, and A + T + in terms of CSF p-tau, AD-related burden, and brain FDG-PET metabolic patterns. However, because of its cross-sectional design, we could not investigate temporal connection between different biomarkers alteration or evaluate whether higher CSF Aβ40 levels, along with pathological CSF Aβ42, may affect the rapidity of cognitive decline.To our knowledge, this study is the first to directly compare CSFAβ42 ratio—and to assess any causal connections between the alteration of Aβ42, Aβ40, and p-tau in the CSF and parenchymal amyloid and tau deposition. Finally, extending the analysis by comparing blood-based and CSF biomarkers could be useful to confirm these relationships and to assess their extensibility for diagnostic and prognostic purposes.Furthermore, fluid biomarkers, represented on a continuous scale, are commonly used in a dichotomous way, applying cut-offs whose thresholds become crucial in clinical practice. Such thresholds are not always available for every biomarker measure, and a global effort of standardization is needed to increase accuracy and reproducibility of these findings. Further investigations using amyloid-PET or tau-PET would be useful to verify our results on the prevalence of pathological AD-related burden across patient groups—which we evaluated indirectly via the CSF p-tau/AβFluid biomarkers are nowadays being introduced into clinical routine practice, positing challenges on their correct use for either supporting or excluding AD diagnosis.42 without pathological amyR nor increased p-tau levels could, under certain circumstances, be essentially considered non-AD, but this conclusion can be hard to reach when clinical presentation and FDG-PET pattern are highly suggestive of AD. Our results suggest that decreased CSF Aβ42 alone could detect Alzheimer’s pathology, since these patients are distinguished from controls by reduced FDG uptake in brain regions typical of AD, and some of them also show pathological measures of AD-related burden.Patients with cognitive symptoms and decreased CSF Aβ40 levels, traced by pathological amyR, associates with higher levels of soluble hyperphosphorylated tau, higher AD-related burden, and a widespread pattern of FDG hypometabolism.On the other hand, the increase in CSF AβConsidering the many experimental novel drugs that see Aβ and tau proteins as therapeutic targets, the specific meaning of different amyloid CSF biomarkers should be considered to support a more accurate selection of patients and search for the best time window for treatment.Additional file 1. Flowcharts summarizing patients’ enrolment (A) and control group selection (B) procedures for the CSF study.Additional file 2. Flowcharts summarizing patients’ enrolment (A) and control group selection (B) procedures for the FDG-PET substudy.Additional file 3. Scatter Plots showing correlations (Spearman’s rho) between CSF p-tau and different amyloid biomarkers  in A-T-, CSFAβ42+/amyR-, CSFAβ42+/amyR+ and A+T+.Additional file 4. Demographical, clinical and biomarkers data from FDG-PET substudy.Additional file 5. Numerical results of SPM comparisons of FDG uptake in CG vs. CSFAβ42+/amyR-, CG vs. CSFAβ42+/amyR+ and CG vs. A+T+.


The aim of the present study was to investigate the behavioral effects of the benzodiazepine midazolam in male mice, in models of anxiolysis, learning, and abuse-related effects.In a first set of experiments, male Swiss mice were submitted to the training session of a discriminative avoidance (DA) task on the elevated plus maze to evaluate anxiety-like behavior and learning after vehicle or midazolam  administration. The same animals were submitted to a conditioned place preference (CPP) protocol with midazolam . In a second experiment, outbred (Swiss) and inbred (C57BL/6) male mice were submitted to a two-bottle choice (TBC) oral midazolam drinking procedure. Animals were exposed to one sucrose bottle and one midazolam  plus sucrose bottle.via the CPP procedure and choice via the TBC procedure showed notable variability. A 2-step cluster analysis for the CPP data showed that midazolam data were well-fitted to 2 separate clusters (preference vs. aversion), albeit with the majority of mice showing preference (75%). Correlational and regression analyses showed no relationship between midazolam reward and anxiolytic-like effects (time spent in the open arms in the DA test) or learning/memory. Two-step cluster analysis of the TBC data also demonstrated that, regardless of strain, mice overall fell into two clusters identified as midazolam-preferring or midazolam-avoiding groups. Both midazolam preference and avoidance were concentration-dependent in a subset of mice.Midazolam (1 and 2 mg/kg) induced anxiolytic-like effects, and all doses of midazolam prevented animals from learning to avoid the aversive closed arm during the DA training session. Assessment of midazolam reward Our findings show that midazolam preference is a multifactorial behavior, and is not dependent solely on the emergence of therapeutic (anxiolytic-like) effects, learning impairments, or on genetic factors . Benzodiazepines are among the most widely prescribed psychiatric medications, with more than 8% of the adult U.S. population reporting benzodiazepine use . This wiDecades of research have helped elucidate the pharmacological mechanisms underlying the behavioral effects of benzodiazepines . HoweverIn addition to these inconsistencies, the relationship between the anxiety-decreasing (anxiolytic) effects and the abuse potential of benzodiazepines remains poorly understood. Anxiety is a risk factor for sedative, hypnotic, or anxiolytic use disorder , and stuThe aim of the present study was to investigate the behavioral effects of the benzodiazepine midazolam in male mice, with a focus on its rewarding effects and self-administration. The rewarding effects of midazolam were evaluated using CPP, and were compared with the anxiolytic effects of this drug using an elevated plus maze-discriminative avoidance task . Self-adad libitum throughout the experiments. The TBC experiments were performed at UESC (Swiss mice) and at the University of Mississippi Medical Center , and subjects were individually housed in polypropylene caging (30 × 19 × 13 cm) with food available ad libitum and fluids restricted to those available within the context of the experiment 24 h per day. All animals were maintained under controlled temperature (22–23°C) and light  conditions.Three-month-old Swiss male mice from the breeding colony of Universidade Estadual de Santa Cruz (UESC) and 3-month-old C57BL/6 male mice obtained from Harlan/Envigo were used. The first set of experiments  was performed at UESC, and animals were group housed (8 per cage) in polypropylene cages (41 × 34 × 16.5 cm). Rodent chow  and water were available Animals were maintained according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals  and in accordance with the Brazilian Law for Procedures for Animal Scientific Use (#11794/2008). The Institutional Animal Care and Use Committees of UESC (protocol #006/2017) and UMMC (protocol #1395) approved the experimental procedures.®) was diluted in sterile saline, and administered intragastrically (gavage) at a volume of 10 ml/kg. For the TBC experiments, midazolam (Roche®) was diluted in a 4% sucrose solution in water to various concentrations (0.008–0.032 mg/mL).For the discriminative avoidance and CPP experiments, midazolam , one of which is aversive , and two adjacent open arms (28.5 x 7 cm) lacking the light and noise stimuli. For this experiment, a test session is performed 24 h after a training session. During the 10-min training session, animals are placed individually in the center of the apparatus with free access to the 4 arms. The aversive stimuli are activated each time the animal entered the aversive closed arm, and were interrupted when the animal leaves this compartment. The test session lasted 3 min, during which the animal was again placed in the center of the apparatus. However, during the test session the aversive stimuli were not activated when the animal entered the aversive closed arm, although the inactive lamp was still present over the closed aversive arm as an environmental cue.® software . Immobility was calculated as the total time spent immobile during the training session. Learning and memory were assessed by quantifying the difference in the percentage of time spent in the aversive closed arm compared to the non-aversive closed arm during the training and test sessions, respectively. Anxiolytic-like effects were measured by the percentage of time spent in the open arms of the device during the training session (longer time spent in the open arms = decrease in anxiety-like behavior). Percent time spent in the open arms was calculated according to the equation: time spent in the open arms / total session time * 100. Percent time spent in the closed arms was calculated according to the equation: time spent in the arm of interest / total time spent in the closed arms * 100.All experimental sessions were filmed for later quantification of the time spent in each of the arms of the device , as well as immobility time (indirect measure of sedative-motor effects) using the ANY-maze® software . The CPP procedure consisted of the following phases:The CPP apparatus consisted of two conditioning compartments of equal size (40 × 20 × 20 cm): compartment A, with black and white vertical lines on the walls and a black wooden floor, and compartment B, with black and white horizontal lines on the walls and a dark (red) smooth floor. The two main compartments were connected by a central compartment (40 × 10 × 15 cm) that was accessible by sliding doors. Test sessions were filmed, and the time spent in each compartment was measured using the ANY-mazeHabituation (Day 1): Animals were placed in the center of the apparatus with free access to all compartments for 10 min. No treatments were administered.Pre-conditioning test (Day 2): Animals were placed in the center of the apparatus with free access to all compartments, and behavior was recorded for 15 min. No treatments were administered.Conditioning (Days 3–14): An unbiased design was used because animals showed no preference for either of the compartments in the pre-conditioning test. Therefore, animals were randomly assigned to an experimental group and to a “midazolam-paired compartment” in a counterbalanced manner. The conditioning sessions were performed during 12 consecutive days, during which the doors remained closed and animals were confined to one of the conditioning compartments. On odd days, animals received an intragastric administration of midazolam. On even days, animals received an intragastric administration of saline. Ten minutes after midazolam or saline administrations, animals were confined to the assigned drug- or saline-paired compartment for 10 min.Post-conditioning test (Day 15): Animals were placed in the center of the apparatus with free access to all compartments, and behavior was recorded for 15 min. No treatments were administered.Subjects were initially habituated to two 15 ml bottles of water for 3 days, followed by habituation to two 15 ml bottles containing 4% sucrose for another 3 days. Following this initial habituation phase, subjects had 24-h access to two 15 ml drinking bottles in their individual home cages, one containing 4% sucrose, and the other containing 4% sucrose plus midazolam . All subjects were exposed to each concentration of midazolam for 14 days, and bottle sides were switched every 7 days. Consumption from each bottle was measured once every 24 h, at which time all subjects were weighed and bottles were refilled. In order to ensure data were not affected by liquid loss due to bottle leaks, for each cohort (Swiss vs. C57BL/6 cohorts) two bottles were left in an empty cage for 1 week, during which time liquid loss was measured and found to be <0.1 ml/day.n = 18) or midazolam at doses of 1 , 2  or 5  mg/kg. Ten minutes after administration, animals were placed individually in the center of the apparatus and had free access to all arms of the apparatus for 10 min. Twenty-four hours after the training session, all animals were submitted to a 3-min drug-free test session.Forty-eight Swiss male mice were randomly distributed into four groups and submitted to the elevated plus maze-discriminative avoidance task procedure, as described previously. On the training day, animals received an intragastric administration (gavage) of vehicle solution (n = 12 per group) on odd days and were confined to the drug-paired compartment for 10 minutes. On even days, all animals received intragastric administration (gavage) of saline and were confined to the opposite compartment for 10 min.One week after the test day, the animals treated with midazolam during the discriminative avoidance task experiment were submitted to the CPP protocol, as described previously. Animals were maintained in the same midazolam groups, receiving the same dose of midazolam during the discriminative avoidance and the CPP protocols. Animals were submitted to the habituation and pre-conditioning test sessions. During the conditioning phase, animals received intragastric administration (gavage) of midazolam . A longer time spent in the compartment associated with the drug compared to the compartment paired with saline (positive score) was considered as indicative of the development of midazolam-induced CPP, while a negative score indicated midazolam-induced place aversion. A score of 0 indicates no preference.* 100.Twenty-nine Swiss (outbred) male mice and 43 C57BL/6 (inbred) male mice were submitted to the habituation and TBC protocols, as previously described. Consumption of sucrose and midazolam plus sucrose solutions were averaged for the last 3 days of self-administration of each midazolam concentration . Preference or aversion for the midazolam bottle over the sucrose only bottle was assessed for each midazolam concentration by calculating the consumption of the midazolam bottle / total consumption of the two bottles t-tests were used as the post-hoc test. In addition to the dependent measures listed above, three derived scores were calculated, including: (1) CPP score = time spent in the drug-paired compartment–time spent in the non-drug-paired compartment during the post-conditioning test session; (2) Learning score = time spent in the non-aversive closed arm–time spent in the aversive closed arm during the EPM training session; and (3) Memory score = time spent in non-aversive closed arm–time spent in the aversive closed arm during the EPM test session (24 h after training). The behavioral data from each experiment were analyzed using one- or two-way analysis of variance (ANOVA), with or without repeated measures . For all analyses, Bonferroni t-tests were used for multiple comparison tests. These analyses, as well as all graphical representations, were performed using the GraphPad Prism software (version 9).The behavioral data from each experiment  were analyzed using one- or two-way analysis of variance (ANOVA), with or without repeated measures . For all analyses, Bonferroni Initial analyses of the data for the CPP studies revealed considerable variance for the CPP score, with distributions of scores predominantly positive  but with negative scores  of relatively high magnitude. We proposed that mice showed either a significant preference or aversion to the midazolam-paired chamber, which represent diametrically opposed predictions for a drug reported consistently to have rewarding effects. Similarly, the TBC studies showed considerable variance for the percentage of preference for the midazolam-containing bottle, leading to a related prediction that mice either preferred or avoided consumption of midazolam. To test these possibilities, we conducted 2-step cluster analysis using CPP score and TBC preference measures. Two-step cluster analysis is a hybrid approach that first calculates a distance measure (centroids) to separate groups, followed by a probabilistic approach to choose an optimal subgroup , 37. Dist-tests, Fisher's exact tests , ANOVA and planned Bonferroni t-tests (to test for dose-associated effects), as well as conducting repeated clustering (n = 15) with newly randomized order of data for each analysis (cluster results can depend on order of data entered). External validation presented more difficulties, because of the lack of available data and analytic approaches providing construct validity associating either concurrent or mechanistic measures of midazolam reward. This study tested two hypotheses that addressed external validation: Midazolam reward is mediated by (1) reduction of anxiety (anxiolysis) and (2) associative learning and memory processes. To assess these hypotheses, correlation (Pearson r) and regression analysis were performed with CPP Score as a predictor of time in open arm (anxiolysis), learning and memory score. In addition, concepts of preference and aversion in CPP procedures are conceptually related to preference and avoidance in the TBC procedure, although as with our hypotheses, there are no available data to address these comparisons directly. Regardless, a general concordance between CPP and TBC with regards to number of clusters  would provide external validation. Cluster analyses were performed using IBM SPSS Statistics software (version 28). For all analyses, family-wise error rate  was constrained to p ≤ 0.05.Cohesion and separation of clusters was evaluated using the silhouette coefficient. Internal validity was evaluated further by comparing clusters using unpaired  = 4.792; p < 0.01]. The two lowest doses of midazolam  significantly increased the % time spent in the open arms compared to vehicle. One-way ANOVA of immobility time also showed a statistically significant difference between groups , with Bonferroni tests showing a significant increase in immobility time for the two highest doses of midazolam  compared to vehicle. A two-way repeated measures ANOVA showed a significant interaction between compartment (aversive closed arm vs. non-aversive closed arm) and treatment (vehicle vs. midazolam) for the % time spent in the closed arms . Bonferroni post-hoc analyses showed that only the vehicle group spent a significantly greater % time in the non-aversive closed arm compared to the aversive closed arm (p < 0.001), demonstrating that animals learned to avoid the aversive closed arm. No significant differences were observed in the % time spent in the closed arms for the groups treated with the lowest doses of midazolam (1 and 2 mg/kg), and animals treated with the highest dose of midazolam (5 mg/kg) spent a significantly lower % time in the non-aversive closed arm compared to the aversive closed arm (p < 0.01), suggesting that all doses of midazolam impaired learning. In agreement, animals treated with 2 and 5 mg/kg midazolam were also significantly different from the vehicle group for both % time spent in the closed aversive arm  and % time spent in the non-aversive closed arm .Results from the training session of the discriminative avoidance experiment are illustrated in  = 3.968; p < 0.05]. Only the vehicle group spent a significantly greater % time in the non-aversive closed arm compared to the aversive closed arm (p < 0.01), indicating that animals learned the association between aversive and non-aversive arms during the training session. No significant differences were observed between the % time spent in the aversive vs. non-aversive closed arms for midazolam-treated animals. Animals treated with midazolam spent a significantly higher % time in the aversive closed arm (p < 0.05 for 2 mg/kg) and a significantly lower % time in the non-aversive closed arm (p < 0.05 for 2 and 5 mg/kg) compared to the vehicle group.Results from the memory measures during the test session of the discriminative avoidance experiment are illustrated in  = 0.593, p = 0.558].CPP results, measured as the difference in time spent in the midazolam- and saline-paired side (CPP score), are shown as a function of pre-conditioning and post-conditioning in Because the CPP score represents a dichotomous variable, with positive numbers indicating preference and negative numbers indicating aversion, we explored the possibility of mice falling into distinct categories. Two-step clustering analysis was conducted separately for each dose of midazolam . Schwarz Bayesian Criterion (BIC) reached acceptable clustering with two centroids for all three doses.  = 1.469, p = 0.293; dose x conditioning phase interaction: F = 0.677, p = 0.539] but a significant main effect of conditioning phase , indicating that a significant aversion occurred, irrespective of dose. To test specifically for dose-related effects, Bonferroni t-tests were conducted within the doses pre- and post-conditioning; and no significant differences were evident (p's > 0.05).We performed additional internal validation tests that also provided information on dose-dependency . For the = 2.118, p = 0.143; dose x conditioning phase interaction: F = 0.672, p = 0.520] but a significant main effect of conditioning phase . Planned Bonferroni t-tests showed that an increase in time spent in the drug-paired side occurred for the 2.0 and 5.0 mg/kg doses of midazolam , indicating significant dose-dependent CPP with midazolam for cluster 2.For cluster 2 . Correlation matrices (Pearson r) are shown in To test specifically if CPP score was a reliable predictor of EPM-DA parameters, individual linear regression analyses were conducted for the cluster 2 (preference) mice . In ever = 4.383, p = 0.030]; however, no multiple comparisons were significant . For C57BL/6 mice, the overall ANOVA was not significant . However, as with CPP in Swiss mice, midazolam preference was highly variable, with mice showing both preferences (above 50%) and avoidance (below 50%). We used 2-step cluster analyses to evaluate the extent to which mice in these studies could be parsed into those that preferred midazolam above 50% levels and those that avoided consuming the drug. For Swiss mice, all three concentrations resulted in 2 clusters (t-tests . For all three doses, the silhouette scores were in the “good” range  = 113.4, p < 0.0001] and multiple comparisons confirmed that all clusters were significantly different from one another . As with the Swiss mice, the two higher concentrations resulted in clustering into two groups that were nearly evenly distributed: 0.016 mg/ml, cluster 1 (preference) = 41.9%, cluster 2 (avoidance) = 58.1%; 0.032 mg/ml, cluster 1 (preference) = 58.1%, cluster 2 (avoidance) = 41.9%. Statistical comparisons verified the differences: 0.016 mg/ml, t(41) = 10.04, p < 0.0001; 0.032 mg/ml, t(41) = 10.98, p < 0.0001. In addition, silhouette scores for all three analyses fell between 0.5 and 1.0, indicating good cohesion and separation of clusters . Interestingly, for C57BL/6 mice, cluster 1 (preference) mice were predominantly in the mixed category, whereas cluster 2 (avoidance) mice were infrequently coded as mixed . This analysis indicates that for Swiss TBC results, effects dependent on dose accounted for half of the subjects in both clusters, whereas with C57BL/6 mice, effects were dependent on dose predominantly for the mice showing preference for the midazolam solutions.A noteworthy characteristic of the TBC data is the observation that different mice were in different clusters across the concentrations. This is expected, because many mice showed mixtures of preference and avoidance depending on the concentration, as is a fundamental characteristic of self-administration data over all drug classes and most procedures. To quantify this phenomenon, we coded each mouse as “same” of “mixed” effects. “Same” indicated a mouse for which all three concentrations were either above 50% preference or ≤50% preference. “Mixed” indicated a mouse for which at least one concentration differed from the other concentrations. For example, a mouse with preference above 50% preference for 0.016 mg/ml but below 50% preference for the other two concentrations was coded as “mixed.” For the two strains, we compared the two clusters by conducted Fisher's exact tests. As shown in Despite the clear and growing concern over benzodiazepine misuse, investigating the abuse-related effects of these drugs has not been as straightforward as researchers might expect. The pre-clinical literature on the effects of benzodiazepines in animal models has been filled with contradictory findings, and establishing models to investigate benzodiazepine reward and reinforcement has been a challenge. Specifically, studies have shown opposite effects for benzodiazepine self-administration –9, benzovia the CPP procedure and choice via the TBC procedure showed notable variability, with evidence that mice developed CPPs or conditioned place aversions (CPAs) with midazolam exposure and, similarly, preferred or avoided midazolam in the TBC model. We evaluated the extent to which mice could be divided into broadly different categories, i.e., midazolam-preferring vs. midazolam non-preferring, using 2-step cluster analysis. This approach was used because it does not require a priori choice of number of possible clusters, opening up the possibility for identification of other sub-groups . Regarding the CPP data, midazolam data were well-fitted to 2 separate clusters, albeit with the majority of mice showing preference (75%). When analyzed separately, the 2.0 and 5.0 mg/kg doses engendered significant preference in the preference cluster, whereas the aversion cluster generally showed robust aversions with no dose-related effects. The majority of mice demonstrated CPP by an increase in time spent in the midazolam-paired compartment, mostly by increasing time spent in the particular chamber vs. shifting preference from one chamber to another. This latter observation suggests that mice already showing an aversion to a drug-paired chamber may not be likely to change to a preference, however, the mice in the aversion cluster mostly showed increased time in the non-drug-paired side instead of no change from pre-conditioning tests.In the present study, assessment of midazolam reward The distinct clusters observed for the CPP experiment allowed us to assess the relationship between reward and other characteristic effects of benzodiazepines. In this regard, midazolam had anxiolytic-like effects in mice, increasing the time spent in the open arms of the modified EPM apparatus, consistent with previous studies , 39. To The finding that midazolam impaired learning and memory of a discriminative avoidance task is consistent with previous pre-clinical studies with midazolam  and otheper se.In addition to testing the hypotheses that midazolam reward is associated with its anxiolytic and cognitive-impairing effects, these comparisons potentially provided tests of external validity for the 2-step cluster approach. Clearly these findings did not provide external support for the clustering, with lack of a relationship between CPP and cognitive effects perhaps the most perplexing. However, it is critical to note that the effects of midazolam in the learning and memory components of the discriminative avoidance task were to impair these processes, whereas CPP and CPA involve forming associative pairings. Moreover, learning to avoid an open arm may represent a form of fear conditioning, as opposed to reward learning represented by CPP, which was the result of 75% of the mice, and while neural circuits mediating aversive and reward learning may overlap, there likely are distinct functional differences . RegardExternal validation of mice being categorized as midazolam-preferring vs. midazolam-averse comes primarily from the TBC experiments. Two-step cluster analysis demonstrated that two different strains of mice overall fell into two clusters identified as midazolam-preferring or midazolam-avoiding groups, with only one exception being the lowest concentration of midazolam tested in C57BL/6 mice, which resulted in an additional (third) cluster characterized as indifference . Both midazolam preference and avoidance were concentration-dependent in a subset of mice, with some showing preference at some concentrations but avoidance at others. However, there was a trend for this pattern to occur more frequently in the midazolam-preferring Swiss mice and a statistically significant difference between midazolam-preferring and midazolam-avoiding C57BL/6 mice, suggesting that mice in the midazolam-avoiding groups tended to only show avoidance regardless of the concentration of drug.Wild type laboratory mice can be divided into two main genetic categories: inbred and outbred . Inbred The present findings corroborate a recent study in non-human primates showing that only half of the subjects self-administered the benzodiazepine alprazolam intravenously, although that study was conducted in rhesus monkeys with a history of opioid self-administration . These fOverall, our findings show that midazolam preference is a multifactorial behavior, and is not dependent solely on the emergence of anxiolytic-like effects, or on genetic factors . Also, the rewarding effects of midazolam in the CPP model emerged even at doses that induced significant learning deficits in mice. The protocols established in the present study can be used in future research to evaluate the neuropharmacological mechanisms involved in the different behavioral effects of benzodiazepine drugs within the same group of animals. Of note, important limitations of our study include the lack of sex differences investigation, with the possibility that different results would have been obtained for female mice. Also, the sample size in our CPP studies limited some of our analyses, and future studies should consider including multiple cohorts of animals to increase sample size in order to better capture benzodiazepine-induced CPP vs. CPA in mice. Regardless, our data emphasize the importance of considering interindividual variability within a sample, and suggest that variability may be an inherent phenomenon to the study of the abuse-related behavioral effects of benzodiazepines. Embracing variability may provide new avenues of study and a better understanding on how and why benzodiazepine drugs are abused.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This animal study was reviewed and approved by the Institutional Animal Care and Use Committees of UESC (protocol #006/2017) and UMMC (protocol #1395).AO-L, EM, JR, and LB were responsible for the study concept and design. CJ-F, MF, BD, YS, NK, TB-S, NJ, IR, and LM contributed to data acquisition. CJ-F, EM-M, AO-L, EM, JR, and LB assisted with data analysis and interpretation of findings. LB and JR drafted the manuscript. All authors agree to be accountable for all aspects of the work, provided critical revision of the manuscript for important intellectual content, and approved the final version for publication.