Context:Aquilariae Lignum Resinatum (ALR), the dry rhizome of Aquilaria agallocha R. (Thymelaeaeeae), has been widely used to treat emesis, stomachache and gastrointestinal dysfunction.Objective: This study evaluates the effects of ALR methanol extract on gastrointestinal motility (GIM) and possible mechanisms of the action involved.Materials and methods:In vivo, the study evaluated the effects of ALR (200–800 mg/kg) on gastric emptying and small intestinal motility in normal and neostigmine-induced adult KM mice. The in vitro effects of ALR (0.2–1.6 mg/mL) on GIM were performed on isolated jejunum of Wistar rats, pretreated with acetylcholine (ACh), KCl, CaCl2, and pre-incubation with l-NAME (a selective inhibitor of the nitric oxide synthase).Results:In vivo, ALR (800 mg/kg) decreased gastric emptying , small intestinal transit . In vitro, ALR concentration dependently decreased the contractions induced by ACh (10−5 M) and KCl (60 mM) with respective EC50 values of 0.35 and 0.32 mg/mL. The Ca2+ concentration–response curves were shifted by ALR to the right, similar to that caused by verapamil (the positive). The spasmolytic activity of ALR was inhibited by pre-incubation with l-NAME.Discussion and conclusions: ALR played a spasmolytic role in GIM, which is probably mediated through inhibition of muscarinic receptors, blockade of Ca2+ influx and NO release. This is the first study presenting a comprehensive description of the effects of ALR on GIM. Functional gastrointestinal (GI) diseases such as functional dyspepsia, irritable bowel syndrome, gastroesophageal reflux, as well as other symptoms  are common and varied pathological illnesses in the GI tract Chang . These dAquilaria agallocha R. (Thymelaeaeeae), recorded in the Chinese Pharmacopoeia  is one of the important drugs for promoting qi circulation and relieving pain in the clinical practice of Traditional Chinese Medicine. Traditionally, it is suggested in prescriptions or used as an herb for the treatment of GI dysfunction such as peptic ulcer, diarrhoea, nausea and vomiting. The plant is also boiled as tea or soaked in wine by the indigenous people, which are thought to be beneficial for the GI tract  were conserved at the School of Pharmaceutical Science and Technology, Tianjin University.in vivo experiments. For in vitro tests, ALR was first ground with Tween-80, the solution was diluted with distilled water so that the final concentration of Tween-80 was 1% in the vehicle. Vehicles used had no effect in control experiments.ALR (100 g) was powdered and extracted with 1 L of methanol for 1 h, three times. The combined methanol extracts were concentrated using a rotary evaporator at reduced pressure and the crude extract was obtained with a yield of 12.18% (w/w). ALR was suspended in 0.5% carboxymethyl cellulose (CMC) for l-NAME and PAG were obtained from Sigma-Aldrich Co. . Acetonitrile, methanol and formic acid were purchased from Merck . HPLC grade water was obtained from a Milli-Q Reagent Water System . Wei-Chang-An pill (WCA) was obtained from Tianjin Lerentang Pharmaceutical Factory . All the other reagents were obtained from Tianjin Jiangtian Chemical Reagent Science and Technology Co., Ltd. . These reagents were of analytical grade. Solutions of all drugs and reagents were dissolved with saline or distilled water, and they were freshly prepared on the day of experiments.ACh was purchased from the National Institute for Control of Pharmaceutical and Biological Products . Neostigmine methylsulphate injection (2 mL:1 mg) and verapamil were donated by Affiliated Hospital of Logistics College of Chinese People’s Armed Police Forces. t = 25 ± 1 °C) and air humidity (60%) controlled room with a 12 h light/dark cycle. They have free accessed to water, but were fasted for 24 h before experiments. The animals were cared for in accordance with the Guide to the Care and Use of Experimental Animals.Adult KM mice (18–22 g) and Wistar rats (280–330 g) were obtained from the Laboratory Animal Center of Health Science, Peking University, Beijing, China (License No. SCXK (Jin)-2009-003). All animals were housed in an environmentally (2) flowrate, 10.0 L/min; drying gas temperature, 360 °C; nebulizer gas pressure, 40 psig; capillary voltage, 4.5 kV; fragmentor voltage, 175 V; skimmer voltage, 65 V; octopole radio frequency voltage, 750 V. The MS spectra were acquired under the positive ion mode. The collision energy was set at 15–40 V and the accurate mass spectra were recorded across the range 100–1700 m/z.The chemical analysis was performed on an Agilent 1290 UHPLC system connected with Agilent 6520 Q-TOF mass spectrometer via an ESI interface . Samples were separated on a reversed phase C18 analytical column . The mobile phase consisted of water–formic acid  (eluent A) and acetonitrile–methanol (50:8) (eluent B), the linear gradient as follows: 0–40 min, 5–100% B; 40–50 min, 100% B; flow rate, 0.5 mL/min. For MS detection, operating conditions were as follows: drying gas , ALR-treated groups  and positive control group  were intragastrically administered in a volume of 0.2 mL/20 g for seven days. Gastric emptying and small intestinal transit rate in 24 h fasted mice were measured using the phenol red method  was added to 4 mL of the supernatant. The absorbance of the sample was read at 560 nm by with the 754 spectrophotometer. In addition, another 10 mice were sacrificed immediately after administration of the test meal, and its absorbance of phenol red is regarded as a standard. Gastric emptying rate was calculated according to the formula: Gastric emptying rate (%) = 1 − (amount of phenol red recovered from test stomach/average amount of phenol red recovered from standard stomach × 100).The amount of phenol red maintained in the stomach was determined by the following methods. The stomach was cut into several pieces and homogenized in 25 mL of 0.1 M NaOH. The suspension was allowed to stand at room temperature for 1 h, and 1 mL of trichloroacetic acid (33% w/v) to 8 mL of the supernatant was added to remove protein. The mixture was centrifuged at 3200×After the excision of the stomach, the length of the whole small intestine was measured from the pyloric sphincter to the ileocecal junction. The intestine was exactly localized by a drop of 0.1 M NaOH to measure the distance travelled by the phenol red. Small intestinal transit rate was expressed as the distance travelled by the test meal relative to the total length of the small intestine , positive control group  or ALR-treated groups . They were administered intragastrically for 30 min prior to intraperitoneal injection of neostigmine (0.1 mg/kg). Phenol red was administered 15 min later and 20 min thereafter, the mice were sacrificed. The gastric emptying and intestinal transit rate were calculated as the method described on the former part.2 1.8, KCl 2.7, MgCl2 1.1, NaHCO3 11.9, NaH2PO4 0.4, and glucose 5.6, pH 7.4). The jejunum tissue was prepared with approximately 2 cm length, and placed in a 10 mL organ bath with Tyrode’s solution, continuously bubbled with a mixture of O2 and CO2 (19:1), and maintained at 37 °C. The resting tension was subjected to 1.5 g and it took 30 min to equilibrate before any drug added.Wistar rats were killed by cervical dislocation after 24 h starvation. The jejunum was removed, flushed, then put into the 0 °C Tyrode’s solution (composition (mM) NaCl 136.9, CaCl−5 M) and KCl (60 mM) were tested  were added sequentially into the bath to examine the effects of ALR on the spontaneous contractions of jejunum strips. In order to investigate prokinetic mechanism preliminarily, the effects of ALR on the contractions of jejunum strips induced by pretreatment with Ach (10+ (60 mM) was used to depolarize the preparations. The jejunum strips were initially stabilized in normal Tyrode’s solution and then replaced with a high-K+ (60 mM) and Ca2+-free solution (composition (mM) KCl 60, NaCl 91.04, MgCl2 1.05, NaHCO3 11.90, NaH2PO4 0.42, glucose 5.55 and EDTA 0.1) for 30 min to remove Ca2+ in the tissues  was added exogenously in a cumulative manner to establish CaCl2 concentration–response curves. The concentration–response curves of CaCl2 were constructed in the absence and the presence of ALR  or verapamil . The contraction induced by 3 × 10−3 M CaCl2 in the absence of ALR and verapamil was considered as 100%.To assess whether the spasmolytic activity of ALR was mediated through the blockade of calcium-channel, high K2S synthetase were used. l-NAME  and PAG  were used to pre-incubate the jejunum tissues for 20 min. Then, the contraction of rat jejunum strips was induced by ACh (10−5 M). ALR  was added sequentially to examine the effects of endogenous NO and H2S on ALR-mediated relaxation. Incubation in the absence of inhibitors was considered as a control group.To analyse the role of gaseous mediator on ALR-mediated relaxation on the contractions of rat-isolated jejunum, inhibitors of NO and Hp Value less than or equal to 0.05 was noted as statistically significant.SPSS17.0 system  was used to analyse the data. All data were expressed as mean ± standard error of mean (S.E.M.) or percentage. Differences among groups were analysed by a one-way analysis of variance (ANOVA) followed by Dunnett’s test. To characterize the chemical constituents in ALR, an UHPLC-Q-TOF-MS method was established. Altogether, a total of 23 compounds were tentatively identified and characterized in ALR, including nine sesquiterpenes (A1–A9), ten 2-(2-phenylethyl) chromone (B1–B9), three flavonoids (C1–C3) and two fatty acids  , Table 1In vivo study, the gastric emptying rate was 62.93 ± 8.74% in normal conditions. ALR at different doses treatment had a little effect on gastric emptying compared with normal mice (p > 0.05). In groups of ALR , the gastric emptying rates were 66.78 ± 7.25%, 64.34 ± 5.47% and 59.74 ± 9.81%, respectively  (ectively . Howevere group) .p < 0.01). In neostigmine-treated mice, neostigmine obviously accelerated the propulsion of the phenol red in the small intestine (85.53 ± 5.57%). The stimulated intestinal transit induced by neostigmine was significantly attenuated by ALR in a dose-dependent manner, with the values of 77.02 ± 6.16%, 73.91 ± 5.96% and 69.60 ± 3.82% (p < 0.05), respectively. The strongest inhibitory activity of ALR was comparable to the positive control   .−5 M) and KCl (60 mM) with respective EC50 values of 0.35 mg/mL and 0.32 mg/mL. The strongest inhibitory effects were 100.00 ± 5.92% and 92.38 ± 3.95%, respectively  treatment. The positive control group  reduced the maximum contraction to 21.68%. The curve of CaCl2 concentration–response in the presence of ALR (1.6 mg/mL) was similar to that of verapamil. They all had significant difference compared with vehicle control (p < 0.01).As the results shown in l-NAME (10−4 M) in a concentration-dependent manner (p < 0.05) (−5 M) did not produce distinct change in the relaxant effect on the relaxation induced by ALR (p > 0.05) (ALR-mediated relaxation of rat-isolated jejunum was significantly reduced by pretreatment with  < 0.05) . However > 0.05) .2+, but also by various neurotransmitters such as ACh, dopamine and 5-hydroxytryptamine (5-HT)  inhibitor that could increase the cholinergic activity in gut wall. The activated cholinergic activity results in the contractile action. The drug is usually used to mimic the general mechanism of action of many prokinetic drugs . The use of isolated tissues permitted us to examine the direct intestinal actions of ALR in the absence of bio-transforming bacteria or metabolic organs. In this study, the experimental models of the spontaneous, ACh and KCl-induced smooth muscle contractions were established. These models were conventional analytical methods of the spasmolytic activity and mechanisms of action of drugs or plant extracts  and KCl (60 mM)  . AccordiM2 and M3. The two receptors mainly serve to produce excitations of GI smooth muscles by parasympathetic neurotransmitter ACh (Ehlert M3 receptor is the main muscarinic receptors which mediated smooth muscle contractions in the GI tract. It causes intracellular Ca2+ mobilization through activation of phospholipase C (PLC)  or release from intracellular stores in the sarcoplasmic reticulum. K+ at high concentrations (80 mM) can induce a depolarization via VDCs. High K+ (>30 mM) is also known to cause smooth muscle contractions through the opening of voltage-dependent l-type Ca2+ channels, thus allowing an influx of extracellular Ca2+ causing a contractile effect  to induce smooth muscle contractions in this study. We found that the KCl-induced contractions of rat-isolated jejunum were effectively inhibited by ALR in a concentration-dependent manner. Substances causing the inhibitory action on high K+-induced contractions are considered to be blockers of calcium influx  ) and PAG  of rat-isolated jejunum were tested. The results showed that relaxation curves of ALR were significantly displaced to the right pretreatment with l-NAME (p < 0.01), but not modified by PAG (p > 0.05). NO acts as a signalling molecule in vascular and GI smooth muscle. It is a vital gasotransmitter in numerous physiological and inflammatory processes  through M2 and M3 receptors via internal and external Ca2+ regulation through G protein pathways in vitro. They also showed that Liriope Platyphylla Wang Et tang dose dependently depolarizes ICC PPTs through M3 receptors via external and internal Ca2+ regulation and via G protein-, a phosphoinositide 3-kinase (PI3K)-, a PLC- and a protein kinase C (PKC)-dependent pathways in vitro. Moreover, they showed that naringenin reduced the amplitudes of the PPs of ICCs in a cGMP/NO-dependent manner by activating Ca2+-sensitive K+ channel  control GIM, which are the pacemaker cells of GI muscularis propria and are involved in the generation of primary electrical pacemaker activity. Kim et al.  found thSo, we suspect that the spasmolytic effects on GIM by ALR is likely related to ICCs. Therefore, further study on the relationship between ALR and ICCs is required.in vivo and in vitro tests were probably attributed to the constituents of the spasmolytic and anti-inflammatory properties, especially for sesquiterpenes and flavonoids. The chemical analysis and the identification of active ingredients further supported the protective effects of ALR on the GIM.Furthermore, phytochemical investigation on the ALR revealed the presence of major active ingredients, including sesquiterpenes, flavonoids and 2-(2-phenylethyl) chromone. These compounds have been reported to exhibit a wide variety of biological effects. It has been demonstrated that some sesquiterpenes of ALR exert antispasmodic, anti-ulcer and anti-inflammatory activities, such as hinesol, nootkanone and champacol identified in this study (Yamahara et al. 2+ channels and regulation of NOS–NO–cGMP pathway, but no participation of H2S. Besides, the effects of ALR on GIM were probably attributed to the spasmolytic and anti-inflammatory properties of active constituents, focusing on sesquiterpenes and flavonoids in ALR. Thus, the use of this medicinal plant in traditional medicine for the treatment of GIM diseases is supported and justified by the pharmacological properties described in this study.In conclusion, the present study suggested that the GI dynamic mechanism of ALR methanol extract was the main inhibitory activity. The antispasmodic activity of ALR was mediated through blockade of muscarinic receptors, Ca


Cryptorchidism refers to an absence of the testis in the scrotal sac. Testicular descent occurs in two stages: transabdominal and gubernacular. The descent of the testis can be arrested in its usual path of descent (true undescended testis) or can migrate from the usual path of descent (ectopic testis). Localising the missing testis is important for surgical planning, as well as for identification of complications that are more common with cryptorchidism. Ultrasound is the initial imaging modality to visualise, as well as localise the testis in cryptorchidism. However, ultrasound imaging is limited in visualising testes that are not superficial in location. This article highlights various examples of abnormal descent of the testis in usual as well as unusual locations and complications of undescended testes. Further evaluation with computed tomography scan or magnetic resonance imaging is needed in indeterminate cases and for identification of complications. We have highlighted the role of specific modalities with imaging findings in this pictorial review for the appropriate selection of each modality in clinical practice. An absence of the testis in the scrotal sac is defined as cryptorchidism. The true undescended testis has arrested migration along its usual path of descent, or it is termed ectopic testis when it migrates from its usual path of descent to lie in an unusual location. An atrophic or congenital absence of the testis may simulate a similar situation. In infants, correct localisation of the testis is essential for surgical management, because the approach may vary with the location. In adults, however, it is still important to localise the testes and identify the complications.1 Most undescended testes migrate into the lower scrotum within the first 3 months of life, probably as a consequence of a postnatal surge of testosterone. Only in less than 1% of the cases does the testis remain persistently undescended by the age of 1 year.1 Cryptorchidism occurs four times more commonly unilaterally than bilaterally.Undescended testes are seen in approximately 1% – 6% of newborn males. The incidence is even higher in preterm infants, reported at 30%.2Around 70% of undescended testes are palpable on clinical examination. Clinically, it can sometimes be difficult to distinguish an undescended testis from a retractile testis, which is excessively mobile.3 The normal course of testicular descent is retroperitoneally from the inferior pole of the kidney to the scrotum.4 The testis shares its embryological development with the kidney, and thus, it initially develops in the upper abdomen and migrates towards the inguinal canal through the deep inguinal ring after 21 weeks of gestation. The migration is almost complete at 30 weeks of gestation. Various factors influence the descent: hormones, including gonadotropins and testosterone, as well as active migration of the gubernaculum, determine its final position. The gubernaculum is the ligament that connects the developing testis to the scrotum and is responsible for inguinoscrotal descent. Under the influence of hormones (e.g. testosterone), the gubernaculum contracts and the testis descends into the scrotum. The gubernacular phase is more prone to error, whereas the transabdominal phase of arrest accounts for 5% – 10% of cases. On imaging, cryptorchidism can be broadly divided into two groups: arrested descent or ectopic testes.In 80% of patients with cryptorchidism, the testis is manually palpable in the inguinal canal.Descent of the testes is along the normal path, but incomplete. The testis may be located in the inguinal canal (80%), near the pubic tubercle or, uncommonly, in the abdomen. The testis is often small and abnormal with a short spermatic cord.6Descent of the testes is away from the normal path. The testis is most often found in the superficial inguinal pouch. Other uncommon locations are perineal, abdominal wall, pelvic, crural, penile and femoral. The testis and spermatic cord are usually normal. Most frequent locations to aid a directed search for the testicles are shown in 5 Starting below the inguinal crease, the common femoral artery and vein can be traced with the ultrasound probe in the transverse axis . The aim is also to detect cases of vanished testis or agenesis. Ultrasound is an initial imaging modality to localise the testes, as well as to assess vascularity on colour Doppler imaging. If the testis is not identified in the scrotum on ultrasound, then the ‘tracking the cord’ technique is useful.rse axis . The spe7 It has superior resolution in demonstrating the superficial location of testes, such as along the rectus sheath, inguinal canal or perineum. Colour Doppler imaging is excellent in demonstrating vascularity within the testes  scans and magnetic resonance imaging (MRI) are used in indeterminate cases on ultrasonography, as well as in the assessment of complications. The testes appear hypodense on CT, and it may be difficult to differentiate them from lymph nodes or small cystic structures. CT scan involves significant radiation and raises concerns regarding the radiation dose as the majority of imaging for cryptorchidism occurs in the paediatric age group. CT scan is, however, superior in evaluating and staging known cases of testicular malignancy.9 Plain and contrast-enhanced Coronal T1-weighted (T1W) MRI can differentiate the gubernaculum, testes and spermatic cord. The spermatic cord can be followed to locate the undescended testes. The ectopic, pelvic or retroperitoneal location of the testes can be easily identified  a testis has not descended, surgery should be performed within the subsequent year, preferably by the age of 18 months. Histological examination proves that undescended testes suffer a progressive loss of germ cells as well as Leydig cells.13 This should, however, take into account the local expertise and practice available. Viable and normal size undescended testes are treated with orchidopexy, depending upon expertise, with laparoscopy or an open surgical approach. A small and atrophic intra-abdominal testis can be treated with orchiectomy.Seventy per cent of undescended testes are palpable manually, and in the remaining 30% of non-palpable undescended testis, the current algorithm is to perform abdominal-scrotal ultrasound first. Ultrasound is sensitive in visualising testes in the inguinoscrotal region or localised to the superficial abdomen wall and perineum. If the ultrasound findings are non-diagnostic, the next imaging modality is MRI. CT is non-invasive but is unreliable in identifying the testes and carries the risk of radiation. As per international guidelines from the American Association of Urology (2014), imaging in cryptorchidism is currently advocated as an adjunct. According to their guidelines, diagnostic laparoscopy or open exploration must be performed on all non-palpable cryptorchid patients.14 It may reduce the extent of exploration and time for anaesthesia. Accurate pre-surgical localisation of the testis can provide the surgeon with the anatomic knowledge to tailor the operative approach. MRI has almost 100% specificity in identifying and localising the testes. Laparoscopy and surgery are difficult in children who have had previous inguinal or scrotal surgery because of scarring, increased risk of injury and reduced mobility of the spermatic cord. Cryptorchidism associated with ambiguous genitalia or hypospadias also need mandatory imaging to evaluate for the presence of Mullerian structures. The authors feel that this topic has not received much attention in the literature recently. This then begs the question that in the absence of visualisation of testes with MRI, is it logical to undergo surgery? Further research into the role of imaging in undescended testis is necessary before concluding imaging just as an adjunct. A simple imaging algorithm for cryptorchidism has been designed by the authors and illustrated in Preoperative localisation of the testis definitely aids in surgical planning and approach.15 The incidence of testicular malignancy among men with an undescended testicle is approximately 1 in 1000–2500, which is certainly higher than the normal population. Testicular malignancy has been reported in 10% – 15% of patients with undescended testes16 (Complications of undescended testes include an increased incidence of infertility, trauma, malignancy, torsion, other associated anomalies, as well as inguinal hernia. Although there has been dispute regarding orchiopexy reducing the risk of testicular cancer, it certainly increases the detection of malignancy through testicular self-examination.testes16  and 9.11 Torsion of an undescended testis can be attributed to the development of a testicular tumour, increasing the weight and distorting the normal anatomy of the organ. Torsion of an intra-abdominal testicle may present as an acute abdomen. Inguinal hernias may be associated with the cryptorchidism17 (If a testicle is located in the inguinal location, it might be prone to trauma or pressure against the pubic bone because of its superficial location . The inchidism17 .Clinicians and radiologists should be aware of the chronology of normal testicular descent, the common and uncommon locations of cryptorchidism, the specific imaging approach, as well as the complications associated with undescended testes.


To determine whether the ratio of cerebrospinal fluid (CSF) immunoglobulin kappa to lambda light chains at time of multiple sclerosis (MS) diagnosis predicts disease progression and whether this was intrinsic to CSF plasmablasts.CSF and peripheral blood were obtained from patients undergoing elective diagnostic lumbar puncture and included clinically isolated syndrome (CIS) (n=43), relapsing remitting MS , primary progressive MS  and other neurological disease controls, both inflammatory  and non-inflammatory . CSF samples were assayed for free and immunoglobulin-associated light chains and on B cells and plasmablasts. Clinical follow-up data were collected during a 5-year follow-up period where available.There was an increased median CSF κ:λ free light chain (FLC) in all MS groups  but not controls . This ratio predicted Expanded Disability Status Scores (EDSS) progression at 5 years, with a lower median EDSS in the group with high (>10) CSF κ:λ FLC . CSF κ:λ FLC correlated with CSF IgG1 κ:λ  and was intrinsic to CSF plasmablasts .These data demonstrate that CSF immunoglobulin κ:λ ratios, determined at the time of diagnostic lumbar puncture, predict MS disease progression and may therefore be useful prognostic markers for early therapeutic stratification. Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system, usually presenting as a clinically isolated syndrome (CIS). Not all patients convert to clinically definite MS, and in those who do the level of disability accumulation is highly variable.All subjects provided written informed consent, in accordance with the Declaration of Helsinki and were not on disease-modifying therapies at the time of sampling and had not received steroids for at least 3 months prior to sampling.et al.Matched peripheral blood and CSF samples were prospectively collected during routine diagnostic lumbar puncture, and blinding was achieved, with all samples analysed before the diagnosis had been formally determined. Samples were collected from patients with MS fulfilling 2010 revisions of the McDonald criteria,10.1136/jnnp-2018-317947.supp1Supplementary file 1g, 8 min). The cell-free CSF was collected and stored at −80°C, and CSF cells were stained for flow cytometry (see below). Peripheral blood was collected into EDTA-containing tubes  and peripheral blood mononuclear cells (PBMC) isolated by density gradient centrifugation .CSF was obtained by non-traumatic lumbar puncture and centrifuged . CSF samples were diluted 1:1 with sample diluent buffer and concentrations calculated from a standard curve.Antibodies specific for human IgA, IgG1-Fc, IgG2-Fc, IgG3 hinge, IgG4-Fc and IgM  were coupled to carboxylated xMAP fluorescent microspheres using the xMAP Antibody Coupling Kit  according to the manufacturer’s instructions. For CSF analysis, all incubation steps were for 30 min at room temperature with agitation followed by two washes in phosphate-buffered saline (PBS). CSF samples (50 µL) were added to a mixture of antibody-coated microspheres (50 µL) and 50 µL assay buffer , followed by 50 µL of biotin goat anti-human κ or λ light chain-specific antibodies , then streptavidin R-phycoerythrin . Samples were analysed using a Luminex-200 and xMAP software, with final concentrations calculated from standard curves.RNA isolation and cDNA synthesis from CSF cell pellets were performed using the μMACS One-step cDNA Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Synthesised cDNA was eluted from the column and collected with 50 µL cDNA elution buffer. Immunoglobulin Kappa Variable (IGKV) and Immunoglobulin Lambda Variable (IGLV) sequencing of cDNA were performed by Adaptive Biotechnologies  using the immunoSEQ survey level assay.Statistical analyses were performed using GraphPad Prism V.7.0 . All data sets were determined to require non-parametric testing following use of the D’Agostino and Pearson normality test and were two tailed.Ig FLC, in addition to OCB, are present in the CSF of patients with MS and are highly sensitive and specific in supporting a diagnosis of MS.The highly variable CSF κ:λ FLC ratio in all MS groups led us to assess whether this ratio might be prognostic for disability accumulation. EDSS were determined at a 5-year follow-up time point following the diagnostic lumbar puncture. We divided the cohort into high (>10) and low (<10) CSF κ:λ FLC ratio groups based on approximately equal numbers in each group. There was a significantly lower EDSS at follow-up in the group of individuals with a high CSF κ:λ FLC ratio, with the lower ratio group accumulating greater disability . There wThere are several possible explanations for the bias towards κFLC in the CSF of patients with MS, unrelated to their antigenic specificity, including assay-dependent sensitivity for either κ or λ, in the FLC and/or intact Ig. We therefore examined the κ and λ light chains present in the intact IgG1, IgG2, IgG3, IgG4, IgA and IgM in CSF of patients with MS. It was clear that the observed bias towards CSF κFLC was recapitulated when examining IgG1κ and IgG1λ, with a strong correlation between the IgG1 κ:λ ratio and FLC κ:λ ratio . There wAlthough the past two decades have seen major advances in the sophistication and availability of diagnostic modalities used in MS (particularly MRI), the prognostic heterogeneity of MS makes temporal disability prediction extremely difficult for any given individual.25et al failed to detect any relationship to disability progression, possibly due to a lower frequency of high CSF κ:λ FLC ratio individuals in their study and/or the shorter follow-up period examined. Our data show that for patients with MS demonstrating a low CSF κ:λ light chain ratio at diagnosis, the combined profile of greater conversion from CIS, higher relapse rates, increased disability progression and greater requirement for therapeutic intervention suggest that this immunological feature of MS is highly predictive of aggressive disease and poor prognosis, although it is worth noting that only disability progression was significantly different.In this study, we demonstrated, for the first time, that the ratio of κ:λ Ig FLC measured at the time of diagnostic lumbar puncture predicts EDSS disability at 5 years postdiagnosis. This is a critical advance on previous studies indicating that the ratio of κ:λ FLC in the CSF might predict the conversion from CIS to CDMS.The presence of B cells and their secreted antibody and FLC in the CSF and brain tissue is well established.34 35The availability of a prognostic marker in the CSF at the time of diagnosis is of clear utility. There are now many highly efficacious therapies for MS, making a rational choice difficult for any individual patient, particularly as some are associated with a significant adverse event profile. MRI protocols are now well established for the diagnosis and therapeutic monitoring of patients with MS. The use of CSF κ and λ assay measurements at diagnosis may help to identify patients with a poor prognosis, justifying the use of highly effective therapies, with potential benefit on long-term outcomes. Larger independent studies would be required to validate the findings and determine the ultimate clinical utility of these measurements.


When given beyond 4.5 h of stroke onset, tissue plasminogen activator (tPA) induces deleterious side effects in the ischemic brain, notably, hemorrhagic transformation (HT). We examined the efficacy of granulocyte-colony stimulating factor (G-CSF) in reducing delayed tPA-induced HT, cerebral infarction, and neurological deficits in a thromboembolic (TE) stroke model, and whether the effects of G-CSF were sustained for longer periods of recovery. After stroke induction, rats were given intravenous saline (control), tPA (10 mg/kg), or G-CSF (300 μg/kg) + tPA 6 h after stroke. We found that G-CSF reduced delayed tPA-associated HT by 47%, decreased infarct volumes by 33%, and improved motor and neurological deficits by 15% and 25%, respectively. It also prevented delayed tPA treatment-induced mortality by 46%. Immunohistochemistry showed 1.5- and 1.8-fold enrichment of the endothelial progenitor cell (EPC) markers CD34+ and VEGFR2 in the ischemic cortex and striatum, respectively, and 1.7- and 2.8-fold increases in the expression of the vasculogenesis marker von Willebrand factor (vWF) in the ischemic cortex and striatum, respectively, in G-CSF-treated rats compared with tPA-treated animals. Flow cytometry revealed increased mobilization of CD34+ cells in the peripheral blood of rats given G-CSF. These results corroborate the efficacy of G-CSF in enhancing the therapeutic time window of tPA for stroke treatment via EPC mobilization and enhancement of vasculogenesis. On average, one American has a stroke every 40 s, and one dies every 4 min . Of the Granulocyte-colony stimulating factor (G-CSF), produced by the bone marrow stromal cells and fibroblasts, stimulates the proliferation, survival, and maturation of cells committed to the neutrophilic granulocyte lineage . ExogenoIn keeping with our goal to pursue the development of G-CSF as a therapeutic agent to expand the limited therapeutic time window of tPA for ischemic stroke treatment, we conducted a follow-up study to examine whether the drug can reduce delayed tPA treatment-induced HT, worsening of neurological outcomes, and mortality in a thromboembolic (TE) model of stroke, a stringent model that closely mimics the clinical situation of vascular occlusion and reperfusion ,15,16,17t (12) = 3.62, p < 0.01), indicating HT occurence (t (12) = 4.03, p < 0.01) (t (12) = 2.67, p < 0.05) (t (12) = 2.21, p < 0.05), indicating a cytoprotective effect of the drug  tPA-induced HT and cerebral infarction in a TE model, we evaluated hemorrhage volume and extent of cerebral infarction 3 days after stroke using spectrophotometric hemoglobin assay and 2,3,5-triphenyltetrazolium chloride (TTC) staining, respectively. Brain sections from the vehicle and G-CSF + tPA groups showed no detectable bleeding A. In conccurence D. Treatm < 0.01) D. Meanwh < 0.05) C,E. Treathe drug E.F  = 35.46, p < 0.001) and day  = 84.62, p < 0.001) effects and an interaction between treatment and days  = 12.05, p < 0.001). Post-hoc testing demonstrated an improvement of stroke-induced motor deficits in G-CSF + tPA-treated rats compared with tPA-treated subjects 7 days after stroke (p < 0.01)  = 304.2, p < 0.001) and day  = 622.4, p < 0.001) effects and an interaction between treatment and days  = 71.72, p < 0.001). Post-hoc tests detected significant improvement of stroke-induced neurological deficits in rats given G-CSF + tPA 7 days after stroke (p < 0.01), compared with rats subjected to tPA treatment only (We conducted elevated body swing test (EBST) and neurological assessments to examine the effects of G-CSF on stroke and on delayed tPA-induced exacerbation of motor and neurological deficits. The tests were carried out prior to stroke induction and at 1 and 7 days after stroke. Two-way ANOVA of the EBST findings showed significant treatment ( < 0.01) A. Analysent only B. In ligent only C. F  = 6.45, p < 0.05) and treatment  = 68.43, p < 0.001), but no interaction between day and treatment. Post-hoc tests revealed higher CD34+ and VEGFR-2 levels in the ipsilateral cortex of G-CSF + tPA-treated animals compared with tPA-treated rats 3 (p < 0.001) and 7 days (p < 0.001) after drug treatment. A significant treatment effect  = 23.83, p < 0.001) was also found in the ipsilateral striatum of G-CSF+tPA-treated animals compared with tPA-treated rats 3 (p < 0.001) and 7 (p < 0.001) days after drug treatment. The von Willebrand factor (vWF) is a distinctive marker for vasculogenesis [F  = 17.42, p < 0.001), and post-hoc tests revealed higher vWF in the ipsilateral cortex of G-CSF+tPA-treated animals versus tPA-treated rats both 3 (p < 0.001) and 7 days (p < 0.001) after drug treatment. A notable treatment effect  = 40.53, p < 0.001) was also found after comparing vWF fluorescence intensities in the ipsilateral striatum between G-CSF + tPA-treated rats and tPA-treated animals 3 (p < 0.001) and 7 days (p < 0.01) after drug treatment. The expression levels of the above-mentioned surface markers in the contralateral side of all rats (stroke and sham) did not vary significantly.The surface markers CD34+ and VEGFR-2 have been used extensively to characterize EPCs ,19,20,21ogenesis ,22. ReprF  = 4.45, p < 0.05), and post-hoc tests revealed higher levels of CD34+ cells in the blood of rats injected with G-CSF + tPA compared to those given tPA only (p < 0.05)  . This reIn accordance with the findings of our previous study , G-CSF aConsidering that up to 80% of human strokes are caused by thrombosis or embolism ,15, we uSome lines of evidence suggest that delayed tPA-induced HT is associated with detrimental effects of tPA on the neurovascular unit, characterized by disruption of the blood brain barrier (BBB), which is mainly comprised of endothelial cells . TherefoVascularization, a reparative mechanism associated with the development of blood vessels and the improvement of tissue microperfusion around the ischemic boundary zone, promotes neuroprotection and improves functional outcomes after stroke ,18. IncrIn summary, using a TE model of stroke, we demonstrated the attenuation of delayed tPA-induced HT and neurological and functional outcome improvements in stroke animals that were administered G-CSF. Our results also indicate a key participation of G-CSF-mobilized EPCs in mediating the above-mentioned effects of G-CSF, consistent with our previous findings in the intraluminal filament stroke model . Moreovehttps://www.nc3rs.org.uk/arrive-guidelines). Male adult Sprague–Dawley rats (approximately 9–10 weeks old) , weighing 200–250 g at the beginning of experiments were used for this study. They were housed in pairs in an AAALAC-approved Research Animal Facility, in a temperature- and humidity-controlled room maintained on 12 h light–dark cycles, with free access to food and water. In order to maintain gender consistency in our long-standing interest over the past two decades in testing stroke therapeutics, the study focused on male rats. Subsequent studies are planned in female rats following the demonstration of treatment safety and efficacy in males.All experiments were performed in accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals, the Guide for the Care and Use of Laboratory Animals, and other approved guidelines of the Loma Linda University Institutional Animal Care and Use Committee . The procedures, data analysis, and reporting were carried out in accordance with the Animal Research: Reporting In Vivo Experiments guidelines  or stroke surgery (see below), and stroke rats were administered intravenously (via the tail vein) vehicle , tPA (10 mg/kg) , or G-CSF (300 µg/kg)  + tPA (10 mg/kg) 6 h post stroke . The ani2, PaCO2, and plasma pH measurements, in our stroke animals, indicating similar degree of stroke insults. Rats that reached the ≥80% CBF reduction during occlusion were used for the present studies.Embolus preparation was performed according to the methods described by Zhang et al. . In brieHemorrhagic transformation was quantified using the spectrophotometric assay of brain hemoglobin content described in our previous study . To measThe animals were subjected to the elevated body swing test (EBST) and neurological examination before (baseline) stroke surgery and then at days 1 or 7 days after stroke. The procedures of the EBST are described in our previous studies ,18. To mThe rats were anesthetized and perfused transcardially with 4% paraformaldehyde in PBS. Their brains were removed and sliced into 40 μm sections in a cryostat and stored at −20 °C. Slide-mounted sections were incubated overnight at 4 °C with antibodies against the endothelial progenitor cell (EPC) markers CD34+  and vascular endothelial growth receptor (VEGFR)-2  or the vasculogenesis marker von Willebrand factor  in PBS. After washing and incubation with secondary antibodies, the sections were washed and incubated for 30 min with Hoecst  at 37 °C. After washing, the sections were mounted on coverslips using Fluoromount mounting medium. Control studies included the exclusion of primary antibodies, substituted with 5% normal goat serum in PBS. No immunoreactivity was observed in these controls.The rats were anesthetized, and blood was collected from their hearts. Thereafter, peripheral blood mononuclear cells were harvested from rat whole blood samples and stained with a PE-conjugated CD34 antibody (Abcam) in PBS for 30 min at 4 °C. The cells were then washed and stained with Fixable Viability eFluor 450  to distinguish between living and dead cells. After washing, the cells were fixed with 1% PFA before analysis using the MACSQuant Analyzer . The appropriate isotype control was used. Data analysis was performed using FlowJo data analysis software .n = 6 or more data points are generally needed for each group in order to obtain reliable results. All data are expressed as mean ± standard error of the mean (S.E.M.). The results were analyzed statistically using one- or two-way ANOVA and subsequent post-hoc Tukey’s tests, or unpaired t-tests when comparing the means of two groups. The data were analyzed by investigators blinded to the experimental treatments. Statistical significance was set at p < 0.05. All statistical analyses were conducted using GraphPad Prism 5 .We determined each sample size by power analysis using a significance level of α = 0.05 with 80% power to detect statistical differences. On the basis of our previous experience with similar experiments ,18, n =


Motivated by the goal of developing new methods to detect early signs of sarcopenia, we investigated if surface electromyographic (SEMG) data recorded during the performance of cyclic, submaximal back extensions are marked by age-specific differences in their time and frequency characteristics. Furthermore, day-to-day retest reliability of the EMG measures was examined.A total of 86 healthy volunteers used a back dynamometer to perform a series of three maximal voluntary contractions (MVC) consisting of isometric back extensions, followed by an isometric back extension at 80% MVC, and finally 25 slow cyclic back extensions at 50% MVC. SEMG data was recorded bilaterally at L1, L2, and L5 from the iliocostalis lumborum, longissimus, and multifidus muscles, respectively. Tests were repeated two days and six weeks later. A linear mixed-effects model with fixed effects “age, sex, test number” and the random effect “person” was performed to investigate age-specific differences in both the initial value and the time-course (as defined by the slope of the regression line) of the root mean square (RMS-SEMG) values and instantaneous median frequency (IMDF-SEMG) values calculated separately for the shortening and lengthening phases of the exercise cycles. Generalizability Theory was used to examine reliability of the EMG measures.Back extensor strength was comparable in younger and older adults. The initial value of RMS-SEMG and IMDF-SEMG as well as the RMS-SEMG time-course did not significantly differ between the two age groups. Conversely, the IMDF-SEMG time-course showed more rapid changes in younger than in older individuals. Absolute and relative reliability of the SEMG time-frequency representations were comparable in older and younger individuals with good to excellent relative reliability but variable absolute reliability levels.The IMDF-SEMG time-course derived from submaximal, cyclic back extension exercises performed at moderate effort showed significant differences in younger vs. older adults even though back extension strength was found to be comparable in the two age groups. We conclude that the SEMG method proposed in this study has great potential to be used as a biomarker to detect early signs of sarcopenic back muscle function. Sarcopenia, the progressive loss of muscle strength and muscle mass with age  is definSarcopenia is diagnosed by muscle function and structure related tests, i.e., muscle mass, hand grip strength and gait velocity , as wellAdopted training protocols that are widely recommended for progressive resistance exercise to novice training seniors , 29 woulMotivated by the goal to develop novel biomarkers that might be used to screen for very early forms of sarcopenia, the primary objective of this research was to examine whether or not age specific effects could be observed in the surface electromyographic representation of healthy individuals engaging in a cyclic back extension exercise. By simultaneously monitoring back muscle activity using these novel SEMG processing techniques, we hypothesize that with the duration of this submaximal cyclic back exercise performed with a load that equated 50% of isometric back extension maximum, the SEMG amplitude would increase to a smaller extent in older than in younger adults. This result is expected because in older adults, the smaller proportion of fast-fatigable motor units requires less fatigue-related compensation; therefore, there would be a smaller increase in net excitatory drive to the alpha motor neuron pool. Likewise, the time-frequency representation of the SEMG data would be less compressed toward the lower frequencies in older as compared to younger individuals, because the proportion of fast-fatiguing muscle fibers is reduced in the muscles of older adults . Due to The study protocol was approved by the Ethics Committee of the City of Vienna - Number: “EK 11–064-VK-NZ” Full address: Ethikkommission der Stadt Wien Magistratsabteilung 15, Gesundheitsdienst der Stadt Wien 3., Thomas-Klestil-Platz 8 Town Town Tel.: 01/4000–87,523. All participants provided written informed consent. The data collection was carried out in accordance with the directives of the Declaration of Helsinki. Participants received financial compensation for their participation in the study.Study volunteers were recruited by word of mouth, by presenting the study to groups of older adults attending institutions with a focus on leisure and social activities in and around the city of Vienna, and by posting flyers in appropriate areas at companies near the Karl-Landsteiner Institute of Outpatient Rehabilitation Research where the data collection took place. A total of 86 asymptomatic volunteers  were enrolled in the study.Subjects were eligible to participate in the study if: they were generally healthy, i.e., were free of co-morbidities ; had no major general health problems that would interfere with testing; were free from any functional limitations ; were independent in their activities of daily life; performed a normal level of physical activity (but did not participate in competitive sports more than two times per week); and, if they were free from any risk factors that could preclude them from participating in vigorous exercise. Physical Medicine & Rehabilitation physicians screened all participants.Volunteers were not considered eligible to participate in the study if: 1) they were unable to follow instructions given in German; 2) experienced more than five mild back or referring back pain episodes  lasting more than two days each within the past year; 3) had a history of spine surgery or any kind of specific diseases of the spine ; 4) were pregnant; 5) complained about any medical condition that might interfere with maximum strength or submaximal endurance testing; or, 6) had a body mass index (BMI) exceeding 35 kg/m2.Participants completed a total of three different tasks as follows: 1) Basic anthropometric measurements and questionnaires that assessed their motivation and physical activity level; 2) warm-up and maximum isometric back extension (MVC) tests; 3) 20 min of rest, in which the surface EMG electrodes were attached; 4) one sustained isometric back extension at 80% of maximum for 30s; and, 5) after a resting interval of approximately five minutes, a dynamic fatigue task performed with a load equating to 50% MVC. All tests were guided by three experienced examiners  and a certified clinical psychologist (BP).The complete set of measurements was repeated in a second experimental session one to two days after the first, and further repeated in a third session six weeks later.A back-extension device  was used to assess maximum isometric back extension torque. The dynamometer is described elsewhere in detail , and conTo allow undisturbed EMG recording from the back-extensor muscles, the sustained 80% MVC, static back extension, and the 50% MVC dynamic exercise were each performed with the “Total Trunk” ; “Total Truck” is purely an exercise, as opposed to a strength test, device. This device is constructed similarly to the F110 extension device , and also consists of a “hip fixation mechanism” like the F110 device. It is, however, equipped with a dorsal sacral pad instead of a back pad. As with the DAVID device, the device has footplates adjustable to the lower leg length, knee pads adjustable to the thigh length, a pelvic belt, and a seat adjustable for height.Patients were seated in an upright position without back support, both feet on the floor, and with both elbows flexed at 90 degrees and the wrists in neutral position according to the manufacturer’s instructions. They then performed a series of three maximum grip strength tests , where the order was alternated between the right and left hand . If the best two tests varied by more than 10%, a further test was performed so that a consistent maximum could be achieved. All values were monitored and recorded.Electromyographic signals were monitored using active double parallel-bar electrode sensors that also integrated triaxial accelerometric sensors . After the skin had been abraded with alcohol and, if necessary, shaved, electrodes were positioned bilaterally over the multifidus muscle at L5, the longissimus at L2, and the iliocostalis lumborum muscles at L1, considering muscle fiber direction and the positioning recommended by the SENIAM project  and by pA triaxial accelerometric sensor integrated into the double parallel-bar EMG electrode sensor  was attached to the lever arm of the dynamometer in a standardized way. It monitored the direction of the trunk movement. By considering the time elapsed for each cycle, mechanic variables like the range of trunk movement, peak, and average movement velocity of the concentric and eccentric bouts of the exercise could be monitored. The triaxial accelerometers acquired pre-amplified signals with a dynamic range of ±1.5 g, a maximum resolution of 0.016 g/bit, and a bandwidth of dc-50 Hz. The accelerometer signals were sampled at 160 Hz with a resolution of 8 bits and EMG Works® acquisition software.Participants completed the International Physical Activity Questionnaire (IPAQ) on each test day . At the After the participants were securely positioned in the F110 device (DAVID®) following the manufacturer’s recommendations, they performed a warm-up at low loads to familiarize themselves with the equipment and test procedures. Thereafter, participants completed two consecutive maximum isometric contractions under supervision. If the second test varied by more than 10%, or if the moment of peak effort was achieved later than 3 s after the onset of the contraction, further trials were permitted until a consistent maximum was achieved. The best score out of two consistent trials was recorded and stored. Verbal instructions and encouragement provided by the testers were standardized and regularly supervised by a clinical psychologist (BP).After the electrodes were attached to the muscles of interest and checked for function, participants were seated on the Total Trunk device using the same positioning variables that were used for the DAVID® device. Participants maintained a 30° ante flexed trunk position for at least 30s with a load equal to 80% MVC that had been derived from the best maximum trunk extension moment  with the F110 DAVID® device. Load was calculated by the mathematical product of the moment recorded by the load cell of the dynamometer and the moment arm defined by the distance of the back restraint and the load cell.Participants performed the cyclic back extension tests in a sitting position with their arms hanging relaxed at either side of their trunk. In a practicing session without external load, participants repetitively flexed and re-extended their trunks between the upright sitting position and 40° forward flexed position following the pace of a metronome and verbal instructions. Participants were also instructed and briefly trained in a standardized breathing technique with inhalation during the eccentric phase and expiration during the concentric phase of the cyclic exercise. Thereafter, participants were required to perform a total of 25 cyclic contractions with the lever arm loaded equal to the 50% MVC. The movement velocity of the flexion-extension cycle was paced with the metronome set at 2 s intervals (2 s extension and 2 s flexion). The qualitative performance of the dynamic contractions was supervised by experienced testers. If the limit of 25 repetitions could not be reached, or the homogeneity of task performance decreased in range of motion (ROM) or movement velocity, the test was stopped early. If the number of successfully performed cycles exceeded 15 repetitions, it was considered for further data processing.Immediately after the dynamic test, participants were asked to rate their back-muscle fatigue level on a BORG visual analogue rating scale. The anchor points of the scale were zero indicating “no fatigue at all” and 10 indicating “the most severe fatigue ever experienced”.MATLAB routines  were used for EMG data processing. All data was preprocessed by skipping the first three seconds of the static task and the first eight seconds (first two cycles) of the dynamic task, and by removing artifacts. EMG data was filtered using a 20 Hz high-pass and 500 Hz low-pass Butterworth filter design. RMS-EMG calculations were performed from the second to the fifth second of the sustained 80% MVC contraction, as described previously . These vEMG signals of the dynamic fatigue task were sequenced according to their concentric and eccentric phases using acceleration data from one electrode positioned on the lever arm of the Total Trunk device. These on-average 1 s EMG intervals corresponded in equal portions to trunk ROM segments during flexion and extension to avoid acceleration, deceleration, and turning phases during the exercise. Thus, movement velocity and muscle length - torque geometry were comparable in the EMG representations not only between the shorting and lengthening of the cyclic exercise but also between the individuals tested  and instantaneous median frequency (IMDF) SEMG variables were computed for all sequenced SEMG intervals recorded during the dynamic shortening and lengthening contractions. SEMG sections, consisting of approximately 2000 samples, were split up into windows of 500 samples. These windows were overlapping by 50%, whereas IMDF and RMS calculations of the first and last 125 samples of the approximately 1 s intervals were rejected to ensure the extraction of the central portion of the angular range of motion. IMDF calculations are based on Cohen Class Time-Frequency representations  for compThe electrical manifestations of muscle fatigue were assessed for each series of contractions by computing the least squares regression line through the 25 IMDF-estimates relative to the 25 signal bursts of each contraction series. The corresponding IMDF SEMG values of one exercise session were averaged to obtain one IMDF value per cycle and phase. Figure Linear regression was applied to the time series of SEMG values to calculate the rate of change in RMS-SEMG and IMDF-SEMG over time; these measures are indicative of muscle fatigue. Following previous recommendations , both thTrunk range of motion was monitored via the sagittal angular displacement as calculated by a geometrical procedure using direction of gravity as reference; this process has previously been described in detail . To calcThe following dependent variables were used in the analysis: MVC as a measure of trunk extensor strength, mean RMS-SEMG and IMDF-SEMG values derived from the initial values of linear regression models, mean RMS-SEMG and IMDF-SEMG slopes normalized by the RMS-SEMG, and IMDF-SEMG initial values. The independent variables were subjects’ age (older or younger than 50 years of age) and sex. All statistical analyses were done in the R environment for statistical computing .For the analysis of age and sex specific differences, biomechanically comparable (velocity and acceleration) dynamic tasks and artefact-free SEMG data from the full set of electrodes was available from 27 older and 29 younger participants  and considered significant if p ≤ 0.01. The sample size estimate for the mixed ANOVAs was calculated with Monte Carlo power simulation [Descriptive statistics served to summarize the participants’ characteristics. To evaluate whether maximum back extension moment or SEMG fatigue variables differed between the two age-specific and sex-specific groups, mixed effects model with “age”, “sex” and “test number” as fixed factors and the random factor “persons” were computed for each outcome variable. mulation  and was mulation , 50. ConGeneralizability Theory (G-Theory) –53 was uOf the 87 participants tested, a total of 56 participants had a fully artefact free EMG data set that included all six electrode-recording sites and was comparable in the biomechanics when performing the task. Of the 31 participants excluded from the analysis, in most of the cases the SEMG signal taken from one side of the pairwise electrodes was contaminated with strong artefacts, usually due to a slight and intermittent electrode detachment during performing the cyclic exercise. Biomechanical inconsistencies due to significant changes in task performance or the early cessation of the fatigue test occurred in three cases, and reasons for exclusion of individual EMG signal recordings are provided in Fig. Participants’ demographic variables “age”, “height”, and “weight”, their functional variables “maximum back extension strength”, “hand grip strength”, and their fatigue ratings at the end of the cyclical exercise did not differ from those individuals who had to be excluded from further analysis Table .Table 1When comparing groups for age specific differences neither maximum back extension strength nor maximum hand grip strength differed significantly. Participants’ total physical activity scores (TPA) were significantly higher in the older than the younger individuals, and their back-muscle fatigue ratings indicated significantly more fatigue at the end of the cyclic exercise in the younger than older participants Table .Of the biomechanical performance variables of the cyclic back extension exercise, the trunk range of motion as well as movement velocity calculated for the concentric and eccentric phases separately were comparable between participants of the two age groups. In addition, these mechanic variables remained constant throughout the dynamic exercise and in each of the two age specific subgroups Table .The normalized RMS-SEMG onsets derived both from the concentric and the eccentric phases of the cyclic exercise did not significantly differ between younger and older individuals, nor between males and females. It is worthwhile to note that the initial RMS-SEMG values would be approximately twofold in size if the concentric portions of the exercise were compared with the eccentric ones.If the concentric portions of the exercise were considered, the RMS-SEMG of all muscles, the longissimus and iliocostalis lumborum recording sites, significantly increased in individuals of both age and sex groups over the course of the exercise routine and did not significantly differ between younger and older participants, nor between males and females. As opposed to the younger participants, no significant RMS-SEMG fatigue increases were observed for the multifidus muscle of the older participants but differences between age groups did not reach a level of significance. For the eccentric portions of the exercise, the RMS-SEMG slopes were found to significantly increase in younger participants in the iliocostalis lumborum electrode recording site. However, neither the respective age nor sex specific differences reached a level of significance. Interestingly, those electrode sites that revealed the most negative IMDF slope showed a significant decrease in the normalized RMS-SEMG changes for the eccentric portions of the exercise, but did not differ significantly between age and sex groups. Tables The IMDF-EMG onsets obtained from the cyclic back exercise tended to be slightly higher in older as compared to younger individuals. Neither age-specific nor sex-specific differences reached a level of significance for both the concentric and eccentric contraction phases.The IMDF-SEMG slopes normalized to the IMDF onset values significantly decreased with the duration of the exercise. Although these slopes were generally more pronounced in younger than in older participants, significant age specific differences were predominantly observed when the IMDF-SEMG slopes were derived from the concentric phases of the exercise. This was true for all electrodes, the pair of electrodes with the most negative slope, and the multifidus and longissimus (in a tendency significant) electrode recording sites. For the eccentric phases of the cyclic exercise, significant age specific differences were observed for the multifidus muscle and the electrode with the steepest IMDF-SEMG slope. No sex specific differences were observed, except for the most negative electrode recording site. There were neither age nor sex specific differences observed for the imbalance scores. The results of the age and sex specific differences of the IMDF-EMG are presented in Table The absolute SEM values of the normalized RMS-SEMG onsets calculated for either the different  recording sites separately, for the most negative electrode, or for all electrodes were found to be generally larger for the concentric than the eccentric phases of the exercise. The highest SEM-values were found in the longissimus and iliocostalis lumborum recording sites and lowest ones if all electrode recording sites were considered. By contrast, the absolute SEM values of the normalized RMS-SEMG slopes were generally smaller for the concentric than for the eccentric phases of the exercise. The highest value was found for the longissimus, and the smallest one when all the electrodes were considered. There were no major differences between the two age specific subgroups.The D-coefficients (a type of ICC) of the initial RMS-SEMG were found to be slightly higher for the concentric phase of the exercise; they ranged between 0.63 (multifidus eccentric) and 0.9 . The respective D-values of the normalized RMS-EMG slopes were again found to be slightly higher for the concentric than the eccentric phases of the contraction with the lowest value observed from the longissimus (eccentric), and the highest value occurred when all the electrodes were considered (concentric). There were no major age specific differences found for these variables. , and the lowest value was found when all electrodes were considered (eccentric). By contrast, the absolute SEM values of the normalized IMDF-SEMG slopes were found to be slightly smaller during the concentric phases of the exercise. The largest value was found for the multifidus (concentric phase), and the smallest one for the iliocostalis lumborum (concentric phase). There were no major differences between the two age specific subgroups.D-values for the IMDF-EMG onsets observed at all the different recording sites exceeded values of 0.8 in both the concentric and eccentric contraction phases, indicating excellent reliability. The respective D-values of the normalized IMDF-EMG slopes were similar amongst the two contraction phases and age groups, with the lowest values observed for the iliocostalis lumborum  and the highest ones for the multifidus , respectively. Such D-values were similar amongst younger and older individuals except for the longissimus (eccentric phase), where younger participants displayed clearly better values than older ones. All the ICC and SEM values calculated for the different EMG recording sites are provided in Table The load/weight administered in this task led to more perceived back muscle fatigue in the younger as compared to older individuals, but was only modest overall;The RMS-SEMG onsets and fatigue related changes did not reveal relevant significant age or sex specific differences;Despite comparable IMDF-SEMG onsets, the respective IMDF-SEMG fatigue related slopes were clearly less pronounced in the older as compared to younger participants, especially when the concentric portion of the cyclic exercise was considered, andBetween days’ re-test reliability of the SEMG variables revealed SEM and D-coefficient values that were comparable between old and young individuals with SEM values indicating low absolute reliability and D-coefficients indicating fair to excellent relative reliability.This study investigated whether the SEMG amplitude and time frequency representations would be sufficiently sensitive to detect age specific differences in healthy individuals engaging in a 50% of maximum, fatiguing cyclic back extensions exercise. The findings of this research revealed that:Overall, the older participants recruited in this study were more physically active than their younger counterparts. In fact, all older individuals clearly exceeded the recommended minimum physical activity levels . Such hiThe biomechanical variables “range of motion” and “mean movement velocity” that had been monitored to control the biomechanical quality of exercise performance were well matched with the overall requirements of the repetitive flexion extension test and kept constant throughout the exercise. Moreover, as neither the onsets nor the changes over time of these variables differed between the younger and older participants in a relevant way, no major confounders on EMG recordings from variable biomechanical performance of the cyclic exercise could be expected. Although the work performed during the exercise was comparable between the two age groups, the younger individuals reported significantly more back muscle fatigue at the end of the cyclic back exercise, even though the respective scores still indicated fatigue to be moderate. This result is inline with those from systematic reviews that found the back muscle endurance function to be better preserved in older as compared to younger individuals , 57. In Research that investigated age specific differences in motor unit control using the needle electromyographic method observed a proportionally higher number of motor units activated that fired at similar  or modesAs was found in previous studies , 62, theIt is interesting that, when the eccentric contraction mode of the exercise was considered, a significant decrease rather than an increase in RMS-SEMG was observed over the course of the exercise for both the elderly and younger group of participants. In eccentric contractions muscle fibers have a greater intrinsic force capacity  and the In static back extensions at increasing strength, the median of the frequency spectrum (MF) only increased up to 30–50% of maximum . If the The only SEMG variable that clearly indicated aging-induced muscle functional changes in cyclically activated back extensor muscles was the IMDF-SEMG fatigue slope. This result corresponds with our previous observation made from static back extensions that had been performed at 80% of maximum for 30s . It is iThe IMDF–SEMG fatigue slopes appeared to be more sensitive to detecting age specific differences during the concentric than in the eccentric phases of the exercise. The SEMG accompanying concentric versus eccentric contraction has been extensively discussed in a recent study . In shorThis study also investigated the reproducibility of the variables of interest and found the relative day-to-day retest reliability for the SEMG variables to be good to excellent, depending on the recording site. Absolute reliability, however, was highly variable for both the normalized slopes and the EMG onset values. This was true for both age specific subgroups. These observations concur with those from earlier research where healthy individuals performed cyclic free lifting  or a susOne may suspect that the fatigue slopes observed from our older population were biased by significantly higher physical activity scores as assessed with the IPAQ and higher than expected back extension strength scores. It is worth noting that the spectral MF-SEMG fatigue method is independent from an individual’s absolute maximum strength score, but the submaximum load needs to be derived from a true maximum performance. Given this, the age specific differences observed with this method would be widely unaffected by the training state of the back muscle unless the respective strength and power scores were considered in addition. This is important because training induced compensation in strength and power decline following an aging related loss of glycolytic type II fibers is mainly achieved through mechanisms leading to an increase in mechanic output in less fatigable type II and fatigue resistant type I muscle fibers , 77. ThiIt could be argued that differences in blood perfusion of the back extensor muscles confounded the findings from the spectral SEMG observed in this study . We did It is also possible that sex specific differences could interfere with the IMDF-SEMG fatigue changes. In fact, fast fatiguing high threshold motor units of the back muscles were found to be smaller in females than males , and forWe attempted to record a full set of EMG data during the cyclic exercise from a total of 87 individuals. Unfortunately, artifact free recording from all electrode sites was feasible in only 56 individuals. Intermittent loss of electrode skin contact leading to major EMG artifacts, which occurred most frequently at the L5 and L2 recording sites, was one of the main reasons for exclusion of data. This may have reduced the statistical power to detect age specific differences in this explorative study. We therefore analyzed the fitted EMG data that considered side related differences in pairwise electrode recordings. Results between primary and secondary analyses were similar. In fact, future research will have to compare different types of electrodes with defined inter-electrode distance that would be unlikely detach during the performance of such a cyclic exercise.Loss of neurons may start in an individual’s later 40s, and seems to be independent from any muscle training performed . As backAmong the different SEMG variables recorded from a cyclic back extension exercise using a load equal to 50% of maximum and performed with a slow movement velocity, the IMDF-SEMG slope demonstrated clear age-specific effects in a reliable way. This result was observable despite the fact that back extension strength was comparable between the two age groups. The RMS-EMG variable appeared less discriminative. Thus, the IMDF-EMG fatigue method possesses great potential for further development toward a feasible biomarker intended to detect early signs of sarcopenic back muscle function.


The goal of this study is to evaluate the potential of scanning acoustic microscopy (SAM) in the determination of atherosclerotic plaques with calcifications by validating this technique with micro-computer tomography (micro-CT), scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). The fibrocalcific plaques were obtained from 12 different patients and initially examined with micro-CT. The images exhibited calcifications within these plaques. For imaging with SAM, approximately 5 μm thick slices were prepared. Sound speed values within calcified regions were measured to be greater than the ones in collagen-rich regions. These fibrocalcific plaques were also examined with SEM and EDS revealing collagen and calcium deposition within these samples. The consistency of the results obtained by all of the modalities involved in our study is an indication of the potential of SAM as a clinical tool for the diagnosis of vulnerable plaques.Microcalcification is the precursor of vulnerability of plaques in humans. Visualization of such small structures  Micro-CT is an accurate imaging modality for the detection of micrometer-sized structures, which provides much higher resolution than cone beam computed tomography (CBCT) does. Micro-CT generally has a resolution of less than 10 μm voxel size, while, the resolution of CBCT ranges between 76 μm to 400 μm7. Micro-CT can perform in-vitro processing of the structure of materials such as composites, polymers, biological materials  and imaging of up to 4 different substances in a material8 using X-rays. Sample preparation and positioning is simple and does not require high vacuum or low temperatures that may adversely affect the structure. On the other hand, micro-CT is an expensive diagnostic imaging tool and also requires an ionizing radiation, therefore, seeking an alternative is inevitable.Microcalcifications or spotty calcifications within the plaques are the indicators of plaque vulnerabilityScanning electron microscopy (SEM) produces images with a resolution in the order of a nanometer, by scanning the surface of the specimen with a focused beam of electrons. Energy dispersive X-ray spectroscopy (EDS) is a chemical characterization technique, which detects all elements ranging from beryllium (Be) to uranium (U) and their distribution within samples, also by the bombardment of the specimen surface with a focused electron beam. EDS can be implemented in electron microscope systems and consequently, both morphological and chemical information about the sample can be obtained. Microcalcification analysis can easily be performed with SEM-EDS; however, this would again be a very expensive method.18 or acoustic impedance of samples20. Two-dimensional maps of an area of around 5 × 5 mm are obtained in a couple of minutes. Similarly, cells and organelles can be examined using higher frequencies of 100 to 1200 MHz28.Scanning acoustic microscopy (SAM), which is a non-invasive and comparably less expensive technique, provides information about the morphology and the mechanical properties of the specimen simultaneously at microscopic levels, rapidly and without a need for special staining. Focused high frequency ultrasound signals are used to calculate either the speed of sound (SOS) through samplesIn our study, we evaluated the ultrastructural properties of the human carotid plaques with scanning acoustic microscopy, micro-computer tomography, scanning electron microscopy and energy dispersive X-ray spectroscopy. The consistency of results obtained by SAM, SEM, EDS and micro-CT is an evidence of the potential of SAM as a diagnostic tool for the determination of microcalcifications in clinics. SAM provided both structural and mechanical information with micrometer resolution about the fibrocalcific plaques and was successful in differentiating the collagen-rich areas from calcified regions. Micro-CT and SEM monitored calcifications, while, EDS provided elemental distribution within samples. Consequently, we can state that SAM is capable of identifying calcifications within the human carotid fibrocalcific plaques, which are signs of plaque vulnerability at the microscopic level and therefore, is a promising tool for the determination of life-threatening plaques in clinics.As can be seen from Fig. μm thick with a microtome and mounted on glass slides for SAM studies. The images obtained using the sound speed mode of SAM were constructed by comparing the reflections of ultrasound signals from front and rear surfaces of the slices. Figure μm.The fibrocalcific plaques, received from patients undergoing carotid endarterectomy operation, were sliced at approximately 5 Table SEM images of the collagen-rich and calcified regions of the plaques were obtained as shown in Fig. Table 32. However, there is a controversy regarding the management of asymptomatic patients, whether to operate, stent or medically follow these cases. There are worldwide many clinics accepting patients, surgical or interventional candidates with high grade carotid artery stenosis, whereas, various others enrolling only symptomatic patients with important carotid artery stenosis. In the recent guidelines carotid endarterectomy or stenting is proposed in patients with high grade carotid stenosis with low periprocedural risks as a class II indication with a level of evidence of B33. In order to be more selective to intervene on asymptomatic patients with carotid stenosis, literature lacks established criteria indicating which patients are prone to stroke in the early or long run. There are studies researching the plaque features34 and various others for prediction of cerebrobascular events35, however, none could be 100% reliable so far. Histopathological structure of atheromatous plaques including carotid plaques has been widely studied. There is sparse information regarding the ultrastructural properties, chemical composition and mineral component of these materials36. A study on lipid-rich plaques of human abdominal aorta was conducted using photoacoustic imaging and the feasibility of visualizing lipid-rich plaques at wavelengths of 970 and 1210 nm was tested37. The photoacoustic spectra obtained in a region within the plaque showed the spectral signature of lipids. In addition, the lipid-rich plaque was imaged successfully, while illuminating the sample through 2.8 mm of blood, demonstrating the possibility of implementing the photoacoustic technique in vivo. In our study, fibrocalcific human carotid plaques were examined in detail with scanning acoustic microscopy (SAM), micro-computer tomography (micro-CT), scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). All of the plaques were fibrocalcific with an insignificant lipid content, therefore, we observed only collagen-rich and calcific regions. As can be seen in Fig. 12. Calcific regions were easily distinguishable, since they were dark-colored, in fact, in some samples they were black due to former harmless thrombosis. The calcified regions of the fibrocalcific plaques were stiff with higher elastic moduli, therefore, sound speed values were calculated to be greater than the ones of the collagen-rich regions. The difference in sound speed values of calcific regions in all samples was due to the difference in calcification amounts, which vary a lot from one patient to the other, even the symptoms are the same. The patients had undergone surgeries after either they experienced a cerebrovascular incident or had 70% and above shrinkage in their internal carotid artery (ICA), without any further information about the history of the pathology of the plaques and their progression. Besides, some patients are asymptomatic and they have been diagnosed by chance. Two dimensional maps of sound speed with a micrometer resolution, as in Fig. 39, and therefore easily distinguishable within tissues like breast or prostate. Doppler ultrasonography has been widely used to characterize carotid plaques by obtaining plaque morphology, which is an indicator of vulnerability40. SAM, on the other hand, can provide morphological and chemical information about the plaques simultaneously, which enables diagnosis of vulnerable plaques with a verification.Carotid endarterectomy is still the gold standard treatment procedure for atherosclerotic carotid artery disease36. In addition, the X-ray diffraction yielded three groups of materials such as predominant hydroxyapatite-type crystals, crystalline materials containing amorphous component and as a last subtype, wholly amorphous material. The most abundant mineral in their atheromatous plaques, as in our materials has been hydroxyapatite. The hydroxyapatite contained crystals of cholesterol and lipid nuclei in different layers, which was a reflection of plaque to be in different stages of its formation. Depending on the SEM-EDS results of our study, the volume difference of calcium and sodium concentrations in arteries with and without atheromatous plaques may be an indicator of relationship in pathologic development of calcium deposits.SEM images were used to differentiate collagen-rich and calcific regions in fibrocalcific plaques and EDS analyses were performed for determining the compositional differences between two regions of interest. In Fig. In this study, we tried to evaluate the potential of SAM in the characterization of fibrocalcific plaques by validating its outcomes with the results of SEM, EDS and micro-CT. The atherosclerotic plaques, before becoming fibrocalcific, are vulnerable and therefore, had a high risk to rupture. Fibrocalcific plaques consist of mainly collagen and calcifications and are said to be stable, however, at the formation stage of microcalcifications, fibrous cap, covering a lipid-rich core, becomes thin and weakened, making the plaque vulnerable. We observed various elemental compositions in forms of hydroxyapatite and calcium oxalate in the extracted calcium plaques of the patients. Therefore, imaging of calcifications at micrometer scale is very substantial. Sound speed maps of the fibrocalcific samples show clearly different values in collagen-rich and calcified regions. Consequently, monitoring capacity of SAM with micrometer resolution strengthen the idea of introducing SAM as an imaging tool for vulnerable plaques.This study was approved by Ethics Committee of Istanbul University Medical Faculty (No: 2018/952). All research was performed in accordance with relevant guidelines/regulations.Patients were enrolled to the study after being informed about the research and following their consent to donate the left minimum amount of blood from the blood drawn to perform routine preparatory laboratory analysis and their carotid plaques, which are collected and histopathologically examined when they were presumed unusual as an institutional health policy. The surgical procedures and sampling were performed by the same surgeon (M.U.) in all the patients using the same standard technique. Blood samples were drawn (5 cc) into dry tube and (2 cc) into ethylenediaminetetraacetic acid (EDTA) tube after insertion of venous line and invasive artery catheter. The operations were performed with deep and/or superficial regional anesthesia. Superficial cervical block was performed with the injection of 10 cc bupivacaine of 0.05%, 6 cc lidocaine of 2% and 4 cc saline combination along the lateral border of the sternocleidomastoid muscle subcutaneously. Deep cervical plexus block was performed at the level of the transverse processes of cervical vertebrae C2, C3, and C4. A combination of lidocaine hydrochloride and bupivacaine hydrochloride was injected after negative aspiration result for blood. A total amount of bupivacaine hydrochloride of 2–3 mg/kg was allowed. Additional prilocaine hydrochloride was used subcutaneously at the incision line as infiltration anesthesia. Additional doses of prilocaine hydrochloride was injected intraoperatively if the patient complained of pain during the procedure. The allowed total dose of prilocaine was 5 mg/kg. Intraoperative remifentanil (0.025–0.05 mg/kg/min) maintained an adequate level of comfort, responsiveness, and cooperation. Continuous infusion of nitroglycerin was used for blood pressure control. Additional diltiazem or metoprolol was administered if needed. After de-clamping, occasionally midazolam was given. Systemic heparin (100 IU/kg) was injected before clamping and was not antagonized after the procedure. A shunt was selectively used in case of neurologic deterioration at cross-clamping test with a duration of 2–3 minutes. A standard incision parallel to the sternocleidomastoid muscle was performed and common carotid artery (CCA), internal carotid artery (ICA) and external carotid artery (ECA) were prepared and dissected. After systemic heparin injection, the arteries were clamped. The consciousness and the neurologic status of the patient were evaluated with patient’s response to verbal stimuli and ability to move contralateral side hand and foot for at least 2–3 minutes prior to arteriotomy. In case of neurologic disturbance, endarterectomy was performed with insertion of a shunt. A longitudinal incision from the common carotid artery to the disease-free level of internal carotid artery was performed. Thromboendarterectomy was performed using an elevator. The excised atherosclerotic plaque was embedded into 2% formaldehyde for experimental investigation. The arteries were reconstructed with patch in all patients. Following hemostasis, a drain was placed to the surgical site and tissues were sutured anatomically. The symptomatic otherwise more stenotic side was prioritized followed by the surgery of the contralateral side, in cases with bilateral carotid disease.μA, 1024 × 1304 resolution, filter of 0.5 mm aluminum, rotation step of 0.9°, and 40 W power. After obtaining micro-focal spot and arranging high-resolution detectors for X-rays, full-scan mode 360° was performed for each plaque in about 50 min scan period and images were created via Ctvox and ctan programs. Reconstruction of 480 raw images in TIFF format was performed using NRecon Software  generating 755 horizontal sections in BMP format.Plaques fixed within 2% formaldehyde were scanned using a Skyscan 1174v2 device  with the following settings: 50 kV, 800 μm thick samples were characterized by scanning acoustic microscope (AMS-50SI), developed by Honda Electronics . Figure μm and a focal length of 1.5 mm, generates single pulses of width of 5 ns with a repetition rate of 10 kHz and also collects the acoustic waves. Water is the coupling medium between the quartz lens and the target. X-Y stage, controlled by a computer, scanned the transducer and therefore the target material. The reflected signals from both front and rear sides of the target were collected by the 80 MHz transducer and analyzed by the oscilloscope. Finally, intensity, sound speed, attenuation and thickness maps of the region of interest with 300 × 300 sampling points were obtained with a lateral resolution of approximately 20 μm.Approximately 5 μm thick plaque sample placed on a flat glass slide. The signal generated by the transducer is transmitted through the coupling medium and focused on the target. Sref is the reflected signal from the glass surface without the target. The reflected signal from the target received by the same transducer is composed of the interference of reflection from front (Ss) and rear (Sd) surfaces of the tissue slice, Ss + Sd. These two components can not be separated in time domain and therefore, intensity and phase spectra are determined by Fourier transformation into frequency domain. The phase of the reflection wave from front surface, ϕfront, and the phase of the reflection wave from rear surface, ϕrear can be written as41f is the frequency of the signal, d is the thickness of the tissue slice, c0 is the sound speed within water and c is the sound speed within the tissue slice. Thickness of the target can be written asFigure Sound speed can be written asAttenuation of ultrasound signal within the tissue can be calculated by dividing the amplitude by thickness.Scanning electron microscopy and energy dispersive X-ray spectroscopy studies were performed in JEOL JIB 4601 Focused Ion Beam Scanning Electron Microscope multi-beam platform coupled with Oxford X-maxN Energy Dispersive X-Ray Spectroscopy system.χ2 test. Pearson correlation analysis was used for the correlation analysis and logistic regression analysis was used for the determination of the impact of variables. Standardized β coefficients and 95% confidence intervals (CI) were calculated. Statistical significance was defined as p < 0.01.Using SPSS for Windows version 19.0 , all statistical analyses were performed. Mean, standard deviation, rate and frequency values were used for the descriptive statistics of the data. The evaluation of the distribution of continuous variables was done by Kolmogorov-Smirnov test. Student’s t-test and Mann-Whitney U test were used for the analysis of parametric and non-parametric data, respectively. Comparison of the categorical variables between groups was done by The small number of the samples is the major limitation of the study. However, similar results obtained with different detailed high technology imaging equipments within the 12 samples has been accepted as sufficient to reach out a conclusion regarding the ultrastructural composition of atheromatous carotid plaques. Another limitation may be accepted as the unavailability of correlation data of plaques in symptomatic and asymptomatic patients, however, a correlation study will be conducted in a larger cohort and presented later on.