The triad of noise-generated, drug-induced, and age-related hearing loss is the major cause of acquired sensorineural hearing loss (ASNHL) in modern society. Although these three forms of hearing loss display similar underlying mechanisms, detailed studies have revealed the presence of sex differences in the auditory system both in human and animal models of ASNHL. However, the sexual dimorphism of hearing varies among noise-induced hearing loss (NIHL), ototoxicity, and age-related hearing loss (ARHL). Importantly, estrogen may play an essential role in modulating the pathophysiological mechanisms in the cochlea and several reports have shown that the effects of hormone replacement therapy on hearing loss are complex. This review will summarize the clinical features of sex differences in ASNHL, compare the animal investigations of cochlear sexual dimorphism in response to the three insults, and address how estrogen affects the auditory organ at molecular levels. Hearing loss is the predominant health issue in recent decades because it places psychological and socioeconomic burdens on the world. According to World Health Organization (WHO), almost 6.1% of the world’s population has disabling hearing loss . Furthermore, it may rise to 630 million by 2030 and over 900 million by 2050 [While the clinical features of noise, drug, and age-related hearing loss had been well understood, recent studies have demonstrated the sex differences of hearing severity in the triad of clinical ASNHL patients and explored the mechanisms underlying the sexual dimorphism in the animal models. This review article will focus on sexual dimorphism in ASNHL from the clinical and basic perspectives, and will explore the available studies to elucidate the effect of sex hormones on the auditory organ.NIHL accounted for at least 16% of all disabling hearing loss and has a demanding societal cost . The patApart from adults, NIHL in adolescents became a popular and crucial issue in recent years  but evidDrug ototoxicity is another main cause of ASNHL in modern society. Abundant evidence has shown that ototoxic agents were mainly transported from the strial vessels or diffused via the round window into the cochlea after intratympanic administration or systemic use ,18,19. VARHL or presbycusis usually represents developing high-frequency hearing impairment and frequently occurs with poor speech discrimination . AccordiIn recent years, a growing literature has shown that hearing loss is a risk factor for dementia . The lesApart from NIHL, ototoxicity, and ARHL, Meniere’s disease and autoimmune inner ear disease are also associated with ASNHL. Meniere’s disease is characterized by fluctuating and progressive sensorineural hearing loss accompanied by episodic vertigo. Although the exact causes of Meniere’s disease are not clear, endolymphatic hydrops are likely causative of this disease . SeveralThe sex differences in ASNHL were also evident in subsequent animal studies ,61,62. SA series of studies in sex differences regarding the mammalian auditory system including mice, rats, chinchillas, rhesus monkeys, spotted hyena, and sheep were conducted ,68,69,70There are sundry sexual facets that contribute to the roles of this dimorphism: genetic factors, anatomical differences, occupation type, employment status, and so on. Some studies reported that males possess a slightly longer cochlear length but this finding still lacked clinical data and pathophysiological evidence ,73. FromAlthough several animal studies have reported the sex difference in NIHL, the results varied in different species or strains. Milon et al. demonstrated that after exposure to 2 h of octave-band noise, female B6CBAF1/J mice had a significantly lower compound threshold shift and reduced permanent threshold shift compared to control male mice. However, no significant difference in hair cell counts and inner hair cell synapse counts between the two groups was noted . AnotherSex differences in ototoxicity are also a widely discussed topic. Various animal models were examined for further investigation due to inconsistent human observational studies as mentioned above. One study provided direct evidence that the female cisplatin group had more deteriorated OAE values than the male cisplatin group among the Wistar albino rats. Although ABR values did not show a significant difference, the female cisplatin group had more apoptotic spiral ganglion neurons . One recRegarding aminoglycosides, one animal study demonstrated that male Long–Evans rats had poor OAE values compared to female rats after treatment with kanamycin . In the Ahl gene was proposed as the reason to explain the trait of C57BL/6J mice in having a different result compared to CBA mice [To understand the mechanism of sexual dimorphism in presbycusis, various animal models were conducted. CBA/CaJ mice experience progressive high-frequency hearing loss first and then gradually experience low-frequency loss. In addition, CBA/CaJ mice do not develop premature hearing loss, thus they are a suitable animal model for evaluating aging hearing ,82. WhenCBA mice ,83. OverCBA mice ,84,85.As the above observational and animal studies have shown that females have better hearing than males in several ASNHL cases, the main sex difference in hearing may attribute to the distinct sex hormones in the respective genders. Among the sex hormones, estrogen is the most important and widely discussed hormone in this field. Androgen is less discussed in the auditory system and its effect on ASNHL is still under investigation.Estrogen plays the most crucial role in this issue. Some animal and human studies have demonstrated that women may be protected against hearing loss because of estrogen and its signaling pathways ,87. SeveEstrogen was regarded as neuroprotective and neurotrophic for the brain and presumably had protective effects on the auditory system . A serieTwo main estrogen receptors (ERs) include estrogen-receptor-α (ERα) and estrogen-receptor-β (ERβ) . Many trIn hair cells, oxidative stress due to overproduction or insufficient detoxification of reactive oxygen species (ROS) during acoustic trauma, ototoxic drug exposure, and aging is the major contributor to cell death in ASNHL . Estroge+ channel is influenced by estrogen levels [The cochlear lateral wall, which consists of a spiral ligament and stria vascularis, is also under estrogen modulation. Stria vascularis is important in the homeostasis of the water and blood circulation in thecochlea. There are several ion channels distributing stria vascularis and the Kn levels . Meanwhin levels ,116. Then levels  because n levels .Another type of orphan nuclear receptors that mimic the ERs is the estrogen-related receptors (ERRs). ERRs have sequence similarities with ERs and share transcription targets with ERs. The ERRs exhibit neuroprotection and modulate cell apoptosis in many target organs via regulating the ROS system or mitochondrial function and may also have roles in maintaining hearing function ,119,120.As estrogen seems to have protective effects on the auditory system in the molecular aspects, it is still unclear whether estrogen could account for the sex differences in the triad of ASNHL. For NIHL, Meltser et al. demonstrated that ERβ knockout mice had temporary hearing loss after acoustic trauma, whereas ERα knockout mice and wild-type mice did not . In addiIn contrast to the possible protective effect of estrogen on the cochlea in NIHL, whether estrogen plays a role in ototoxicity is still under investigation. Low estrogen may be associated with the decreased distortion product OAE and increased ABR thresholds in ovariectomized rats after cisplatin treatment . ConversRegarding ARHL, the fluorescence intensities of ERα and ERβ were decreased in both sexes when aging and elderly female mice still had higher ERβ levels in spiral ganglion cells, vestibular hair cells, dark cells, and vestibular ganglion cells than males. In contrast, ERα level showed a gender difference only in spiral ganglion cells . These fAndrogen is another dominant sex hormone that regulates the development of male characteristics, strengthens muscle mass, and increases energy and libido. Androgen is mainly produced in the testicles in males but a part of androgen is synthesized in the ovaries and adrenal glands . In contThe previous animal study had shown that testosterone in serum increased neural thresholds in females in a frequency-specific way . From a Even though the benefits of hearing protection from ERs were evident, the clinical usage of estrogen to protect or treat ASNHL remains difficult to validate. From a therapeutic perspective, estrogen regulates many physiological functions in the whole-body system such as cardiac, gastrointestinal, nervous, and respiratory systems rather than hearing alone. There is more evidence regarding the interactions between sex hormones and the function of the inner ear, especially in the mechanism of hearing impairment and balance disorders in elderly females and pregnant women . ParticuFor decades, hormone-replacement-therapy (HRT) users are the most available group to evaluate how estrogen influences hearing in the clinical aspect. It is challenging to compare and interpret because HRT regimens vary in dosage, composition, duration, and initiation with regards to the onset of menopause . CurrentAnother case-control study included 124 postmenopausal women and found that progestin (as a component of HRT) resulted in poorer hearing, whereas estrogen alone showed no significant hearing difference but still with a better trend than the non-HRT control . Their gTaken together, estrogen has a positive effect on hearing and has been proved in vitro and in vivo. In clinical practice, estrogen and its derivatives are predominantly used in HRT regimens. However, most HRTs are comprised of progestin and its derivatives which showed negative effects on hearing. This is characterized as the main reason the previous human studies showed inconsistent outcomes. As we are aware that estrogen operates in the hypothalamic–pituitary–gonadal axis, which functions not only in hearing but throughout the whole body, the adequate dose necessary to reach the therapeutic effect and avoid side effects on other organs still needs to be investigated. Therefore, aside from using systemic estrogen as a therapy, localized use may be the better choice to apply estrogen and its derivatives to improve hearing impairment. Some strategies for cochlear drug delivery such as nanoparticles, hydrogels, or micropumps can be the focus of future investigations ,144.Sex differences are important in the studies of translational neuroscience . AlthougLooking ahead, the study of stem cells and gene therapy in recent years provides new directions for the development of ASNHL treatment in the future decades . Althoug


Background and aim: AZOOR is a rare disease characterized by loss of zones of outer retinal function, first described by J Donald Gass in 1993. Symptoms include acute onset photopsias and subjective visual field losses. The syndrome is characterized by a normal fundus appearance, scotomas and electroretinographic changes pointing towards outer retinal dysfunction. Evolution, response to immunosuppressive treatment and outcome are difficult to predict. The aim of this small case series was to identify the morphological changes and sequence of events in AZOOR thanks to multimodal imaging. Methods: Charts of AZOOR patients seen in the Centre for Ophthalmic Specialized care  were analyzed by multimodal imaging including fundus photography, fluorescein angiography (FA), indocyanine green angiography (ICGA), blue light fundus autofluorescence (BL-FAF) and spectral domain optical coherence tomography (SD-OCT) in addition to a complete ophthalmological examination including visual field testing and microperimetry, as well as OCT angiography (OCT-A) and ganglion-cell complex analysis when available. Cases and Results: Three AZOOR patients with a mean follow-up of 47 ± 25.5 months were included following the clinical definitions laid down by J Donald Gass. The primary damage was identified at the level of the photoreceptor outer segments with an intact choriocapillaris and retinal pigment epithelium (RPE) layer, these structures being only secondarily involved with progression of the disease. Conclusion: Although AZOOR has often been included within white dot syndromes, some of which are now known to be choriocapillaris diseases (choriocapillaritis entities), our findings clearly commend to differentiate AZOOR from entities such as MEWDS (Multiple evanescent white dot syndrome), APMPPE , MFC  and others, as the damage to photoreceptors is primary in AZOOR (a retinopathy) and secondary in choriocapillaritis (a choriocapillaropathy). Gass in the Donders lecture in 1992 and published in 1993 . This asA 36-year-old woman was sent for a second opinion. In her past ocular history, she had been treated with systemic corticosteroids for an alleged optic neuritis OD (Oculus dexter). She complained of blurry vision and an annular scotoma OD. The electroretinogram had shown diffuse loss of rod photoreceptors OD and was normal OS (Oculus Sinister).At presentation her visual acuity was 0.8 OD and 1.0 OS (Snellen chart). There was no inflammation on either side and laser flare photometry values were within normal limits showing no subclinical inflammation. Fundus examination on both sides was essentially normal.® visual field testing  showed a ring scotoma OD, and normal OS. Microperimetry was decreased OD (410/560) and within normal limits OS (480/560).OctopusFA showed discreet late ring hyperfluorescence around the fovea OD and at the inferior border of the disc OS and along the superior temporal arcade OS.  ICGA shoBlue light fundus autofluorescence (FAF) showed hyperautofluorescence of the whole posterior pole OD, except for a central macular area that was normal OD . On the In both eyes the areas of hyperautofluorescence corresponded to the loss of the photoreceptor outer segment line on SD-OCT. Taken together, (1) when compared with FA and ICGA, the areas of hyperautofluorescence corresponded in both eyes to discreet late FA hyperfluorescence and to retained ICGA fluorescence, indicating that there was choriocapillaris integrity and perfusion; (2) combined FAF, FA and ICGA hyperfluorescence indicated a decreased masking effect because of altered or absent screen due to damage and/or loss of photopigment following damage/loss of the outer segments of photoreceptors. The patient planned a pregnancy. Therefore, no immunosuppressive therapy was initiated. After six years of follow-up, the situation in the left eye had remained stable. On the right-side visual acuity had decreased to 0.6, the ring scotoma had slightly increased  but the Multimodal imaging findings remained stable in the left eye. On the right side, the peripheral fundus took the aspect of a pseudo retinitis pigmentosa with pigment clumps .FA late hyperfluorescence in the periphery had increased substantially over the whole fundus due to atrophic changes of the RPE/choriocapillaris complex which corresponded to hypofluorescent areas on ICGA.  The patt®, Novartis, Cambridge, MA, USA). One year prior to her visit a scotoma had also appeared in her left eye. Electrophysiology had shown diffuse loss of rod photoreceptors more pronounced in OD than OS.This 56-year-old woman was sent to our center for a scotoma in her right eye that had first been noted 17 years prior to her visit. Nine years earlier the scotoma had increased in OD which led her doctors to discontinue the hydroxy-chloroquine therapy she had been given during 17 years for the treatment of systemic lupus erythematosus. Two years prior to her visit, her doctors had started an immunosuppressive treatment for her systemic disease with mycophenolic acid, 720 mg BID  (Snellen chart). Intraocular pressure was 14 mmHg ODS. There was no anterior inflammation on either side visible at the slit lamp, but laser flare photometry (LFP) showed slight subclinical inflammation with values of 5.8 photons/milliseconds (ph/ms) OD and 8.9 ph/ms OS. Fundus examination showed a pale ring around the fovea OD A and was® visual field testing  showed a scotoma temporal to the fovea OD and normal OS. Microperimetry was within normal limits OD (448/560) and OS (504/560). Ganglion cell layer measurement showed an almost complete loss outside of the fovea temporally. FA showed faint hyperfluorescence corresponding to the C-shaped hyperautofluorescent area on FAF OD (see below). (Octopus below). , FA 1 Wi below). , FA 1 FAFundus autofluorescence (FAF) showed an extensive C-shaped hyperautofluorescent area around the fovea with dots of hypoautofluorescence within this zone along the superior temporal arcade OD. A On the The areas of hyperautofluorescence on FAF, slightly hyperfluorescent on FA and hyperfluorescent on ICGA, indicated that the choriocapillaris was unharmed . SD-OCT showed loss of the photoreceptor outer segments in these areas, explaining the FAF autofluorescence due to the loss of the masking effect of the RPE lipofuscin by the loss of photoreceptor layer. The analysis of the ganglion cell complex by Optovue OCT showed that there was a substantial ganglion cell loss in the areas of photoreceptor loss delineated by the different imaging modalities .  The left®, Novartis, Cambridge, MA, USA), 720 mg BID and infliximab  5 mg/kg every 6 weeks and after 18 months evolution was stable, as far as FAF, FA, ICGA and SD-OCT was concerned OU. , 40 mg every 2 weeks was started and 6 months later Mycophenolate Mofetil , 2 g per day was added. When the disease progressed despite dual immunosuppressive therapy, a second opinion was solicited. She had gone through numerous episodes of tonsillitis and her ASLO titers (antistreptolysins) had been elevated several times.This 44-year-old woman was sent to us for a second advice by her uveitis specialist. She was consulted for left photopsias, decreased contrast sensitivity, photophobia and scotomas without any overt intraocular inflammation. FA showed bilateral retinal vasculitis. SD-OCT showed loss and/or disruption of the photoreceptor outer segment layer, the ICGA was normal—in particular no choriocapillaris circulation voids were detected. A neuroophthalmological cause had been excluded. Electrophysiological investigation detected photoreceptor dysfunction ODS with reduced amplitudes. FAF showed left hyperautofluorescence in the posterior pole and one year later the right eye was involved in a similar fashion. Adalimumab  as a third immunosuppressive agent but the situation slightly deteriorated further at 4 months follow-up.Multimodal imaging analysis of AZOOR identified the outer retina as the structure primarily involved, as already hinted by Gass who, adequately, spoke of an outer retinopathy  Table 2Table 2. OCT angiography did not contribute significant additional information in the two patients in whom it was performed. It simply showed integrity of the choriocapillaris, which was already demonstrated on ICGA .While imaging findings were consistent in all three patients, the evolutionary patterns, difficult to predict, differed in the 3 patients. In our series it went from minimal progression without treatment in patient 1, to clinical control of disease with dual immunosuppressive therapy in patient 2, and to progression despite triple immunosuppressive therapy in patient 3. Disease was bilateral in all 3 patients with subclinical non-progressing evolution in the fellow eye in two patients.AZOOR was included by Gass in the spectrum of “white dot syndromes” including MEWDS, AIBSE, Multifocal choroiditis and others now known to be inflammatory diseases of the choriocapillaris. Reports including AZOOR in this spectrum, lumping it together with choriocapillaritis entities, continued to be published up to recently after Gass put forward this hypothesis ,10,11,12In contrast, in AZOOR the structure primarily affected is the outer retina as was hypothesized by Gass. With the availability of multiple imaging modalities, the physiopathology of the disease, photoreceptor damage, could be precisely demonstrated ,17,18. HOur small case series verified this disease mechanism. Combined analysis using FA, ICGA, BL-FAF and SD-OCT pointed towards the photoreceptors as the site of the primary lesion process while choriocapillaris was preserved and perfused, thus differentiating AZOOR from choriocapillaritis entities. One element that our AZOOR series had in common with choriocapillaritis was a mean myopic refraction of −2.5 diopters.The two types of conditions have been described to occur sometimes in the same patient ,20,21. TAlthough the characterization of typical AZOOR cases is quite precise, atypical forms involving the outer retina can occur, difficult to classify within the well-defined AZOOR criteria.Retinal vasculitis was present in two of our patients, unilateral faint peripheral in one eye in patient 2 and diffuse pronounced and bilateral in patient 3, a finding which has been described previously .Bone spicule pseudo retinitis pigmentosa evolution was noted in one patient with bone spicule lesions after 6 years of evolution in patient 1 which has also been described previously . The mosAs AZOOR is a rare disease, no therapeutic trials have been conducted and treatment is empirical. Spontaneous remission has been described . Favorab


The subjugation strategy employed by the jewel wasp is unique in that it manipulates the behavior of its host, the American cockroach, rather than inducing outright paralysis. Upon envenomation directly into the central complex (CX), a command center in the brain for motor behavior, the stung cockroach initially engages in intense grooming behavior, then falls into a lethargic sleep-like state referred to as hypokinesia. Behavioral changes evoked by the sting are due at least in part to the presence of the neurotransmitter dopamine in the venom. In insects, dopamine receptors are classified as two families, the D1-like and the D2-like receptors. However, specific roles played by dopamine receptor subtypes in venom-induced behavioral manipulation by the jewel wasp remain largely unknown. In the present study, we used a pharmacological approach to investigate roles of D1-like and D2-like receptors in behaviors exhibited by stung cockroaches, focusing on grooming. Specifically, we assessed behavioral outcomes of focal CX injections of dopamine receptor agonists and antagonists. Both specific and non-specific compounds were used. Our results strongly implicate D1-like dopamine receptors in venom-induced grooming. Regarding induction of hypokinesia, our findings demonstrate that dopamine signaling is necessary for induction of long-lasting hypokinesia caused by brain envenomation. Highlighted Article: Subtype-specific dopamine receptors are involved in the manipulation of host behavior by the parasitoid jewel wasp. Ampulex compressa) manipulates its host the American cockroach (Periplaneta americana) through a unique envenomation strategy aimed at hijacking its behavior. This is accomplished through injection of venom directly into cockroach head ganglia  and gnathal ganglion (GNG). In particular, after seizing the cockroach pronotum with its mandibles, the wasp injects its venom into the central complex (CX), a ‘higher center’ known to regulate motor behaviors (Drosophila melanogaster) and honeybee (Apis mellifera), among others ] were raised in plastic containers (50×50×70 cm) at 27°C under a 12 h:12 h light:dark cycle with water and food (cat chow) ad libitum. Wasps [Ampulex compressa (Fabricius 1781)] were raised in Perspex cages (40×50×50 cm) at ambient temperature of 30°C and humidity of 40% under a 12 h:12 h light:dark cycle. They were provided with water and honey ad libitum. All experiments performed comply with Principles of Animal Care  and with the current laws of the State of Israel.Cockroaches [Spontaneous grooming and locomotory behaviors of adult male cockroaches were measured in a circular arena with a radius of 30 cm. Two stopwatches were used to measure duration of spontaneous walking and spontaneous grooming in bouts of 10 min for a total of 30 min. In every experiment, spontaneous behavior was always observed before (baseline) and after treatment. Escape behavior was measured in a circular arena with a total radius of 30 cm divided into sectors with four concentric circles having radii of 5, 15, 25 and 30 cm. Cockroaches were placed in the center of the arena and stimulated with a soft paintbrush. The escape response was calculated by measuring distance traveled in centimeters from the center to the end of a continuous running bout. Escape distance was then binned into circles with radii of 5, 15, 25 and 30 cm. Escape behavior was tested in three trials spaced by 10 min.2) for a few seconds, then placed on a Petri dish covered in clay. The body and antenna were fastened with clay and the neck was gently tied with a thread to reduce hemolymph flow to the head capsule. A small flap in cuticle between the ocelli was cut to expose the brain. A glass capillary injection needle connected to a Nanoject™ (Drummond Scientific) nanoliter injector was then inserted in the center of the brain targeting the CX and 2×9 nl of drug were injected. The small flap of the cuticle was then closed and sealed off with wax.CX injections were performed as described in −1): NaCl 214, KCl 3.1, CaCl2 9, sucrose 50, HEPES buffer 5 , and sliced into 60 µm sections with a vibratome (Leica VT 1000S). Janus Green tracer  was co-injected as a marker to verify accuracy of injection.After behavioral testing, accuracy of injection was verified histologically. First, the cockroach head was removed and fixed overnight in 10% formalin . Then the brain was removed from the head, embedded in 6% agar in saline consisting of the following components  between these two ganglia were crushed. Crushing the CirC removes descending activity from the brain (and the CX) and therefore can be used to test the role of the GNG alone in the venom induced grooming behavior. Procedurally, cockroaches were anesthetized with COAll drugs were injected directly into the CX following the procedure described above under ‘Injection’ above.−1). In the first experiment, cockroaches were divided into three groups of 10 individuals each. In each group, spontaneous behavior was evaluated at different time intervals  after EEDQ injection. In the second experiment, cockroaches injected with EEDQ were divided into three groups, then subjected to a wasp sting 6, 12 and 24 h after injection. The behavioral study was performed immediately after the sting. In the last experiment, cockroach escape behavior was tested 1 h after EEDQ injection. This behavior was then compared with the escape behavior of stung and EEDQ-injected cockroaches.2-Ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline (Sigma), an irreversible broad-spectrum antagonist of D1 and D2 receptors , was inj−1) into the CX immediately prior to EEDQ injection (18 nl EEDQ 0.01 mmol l−1). Cockroach behavior was evaluated 6 h after injection (n=12). Cockroaches were then subjected to the wasp sting and spontaneous behavior  was quantified again.Cis-(Z)-flupenthixol dihydrochloride (Sigma), a reversible antagonist of D1 and D2 dopamine receptors  was inje−7 mol l−1) into the CX (n=12). Spontaneous behavior was evaluated 2 h after injection.(±)-SKF-38393 hydrochloride (Sigma), a specific D1 receptor agonist , was inj−7 mol l−1) into the CX of cockroaches (n=12). Spontaneous behavior was evaluated 2 h after injection.2-Bromo-α-ergocryptine methanesulfonate salt (Sigma), a specific D2 receptor agonist , was inj−1) before EEDQ . In this experiment, spontaneous behavior was evaluated 6 h after injection. Thereafter, cockroaches were subjected to a wasp sting and spontaneous behavior was quantified again.R (+)-SCH-23390 hydrochloride (Sigma), a specific reversible D1 dopamine receptor antagonist  was inje−1) before EEDQ (18 nl EEDQ 0.01 mmol l−1). In this experiment, spontaneous behavior was studied 6 h after injection. Thereafter, cockroaches were subjected to a wasp sting and spontaneous behavior was quantified again.(S)-(−)-Sulpiride (Sigma), a specific reversible D2 dopamine receptor antagonist  was injehttps://www.rstudio.com/). Results are expressed as means±s.e.m. Mean differences between two groups were examined by parametric t-test and paired t-test and by non-parametric Mann–Whitney and Wilcoxon tests.All statistical analyses were performed in RStudio . From this experiment, we conclude that the CX has a major role in the venom-evoked grooming response. Subsequent pharmacological experiments described below targeted this brain region to elucidate involvement of dopamine receptors in the grooming response. In addition, we evaluated the role of dopamine receptors in long-lasting hypokinesia evoked by the wasp sting.Since the wasp injects venom into both head ganglia (the brain and GNG), we first investigated whether the CX of the brain or the GNG of cockroaches triggers the venom-evoked response. As a first approach to this question, circumesophageal connectives (CirC) between the brain and GNG  were crun=12). Thirty minutes after injection, the duration of spontaneous grooming duration increased significantly compared with grooming duration before injection . By contrast, walking duration decreased  compared with walking duration before injection.We tested whether dopamine signaling is involved in spontaneous grooming and walking by injecting selective DAR agonists into the CX . Actionst-test, P<0.01), whereas walking duration decreased drastically . These experiments indicate that D1-like, but not D2-like, receptors drive grooming behavior. In contrast, activation of both receptor subtypes suppresses spontaneous locomotory behavior.In the second experiment, CX injection of bromocriptine, a specific agonist of D2-like receptors, was performed on 12 cockroaches. Thirty minutes after injection, the duration of spontaneous grooming decreased slightly (paired n=10) was measured at three different time points after EEDQ injection: 6, 12 and 24 h, respectively , but spontaneous walking was not affected significantly. Cockroaches observed 12 h after injection presented no significant differences in either spontaneous walking or grooming compared with behavior pre-injection. Observations made 24 h after EEDQ injection revealed a significant increase in spontaneous walking duration , but no difference in grooming duration. From these experiments, we conclude that EEDQ antagonism of DAR suppresses spontaneous grooming for at least 6 h and increases spontaneous walking, but only after a delay of between 12 and 24 h.EEDQ is a broad spectrum irreversible antagonist of both D1-like and D2-like receptors in insects  and vertectively . When exn=30) were subjected to CX injections of EEDQ prior to a sting. Cockroaches were divided into three groups (n=10) according to timing of the sting following EEDQ injection: 6, 12 and 24 h, respectively  compared with non-EEDQ injected stung cockroaches. In contrast, no significant difference in walking duration was observed at this time point. Animals stung 12 h after EEDQ injection also exhibited a highly significant reduction in grooming . Again, spontaneous walking duration in this treated group did not differ from that of non-injected stung cockroaches. Twenty-four hours after EEDQ injection, grooming duration of stung cockroaches did not differ from non-injected stung cockroaches. Likewise, walking duration in these EEDQ-treated animals was not different to that of non-injected stung cockroaches. From these experiments, we conclude that DARs mediate venom-evoked behavior and, in particular, EEDQ antagonism of DARs suppresses grooming behavior typically observed in stung cockroaches 6 h after EEDQ treatment, an effect that dissipates within 12 h.To verify dependence of venom-evoked behaviors on dopamine signaling, cockroaches . From these experiments, we conclude that protection of DARs from irreversible EEDQ block restores venom-evoked grooming, providing further support for the critical involvement of DARs in venom-evoked behavior. However, walking duration is not affected by these treatments.We then asked whether protection of DARs from irreversible block by EEDQ could rescue normally observed venom-evoked grooming. Such protection was provided by pre-treatment of stung, EEDQ-injected cockroaches with the broad spectrum, but reversible DAR antagonist flupenthixol. Indeed, stung cockroaches injected with flupenthixol briefly prior to EEDQ injection showed normal post-sting grooming when stung 6 h after treatment . In contt-test, P<0.001), whereas walking duration was unaffected compared with non-injected stung cockroaches. These experiments provide further evidence that venom-induced grooming behavior is dependent on D1-like receptor signaling.To further investigate roles of D1-like and D2-like receptors in venom-induced behavioral changes, DAR-specific antagonists were injected into the CX of cockroaches prior to the sting . Injectit-test, P<0.001) , which corresponds to a full escape running response . The staP<0.001) . EEDQ alOne hallmark of venom-induced behavioral manipulation of cockroaches stung by the jewel wasp is intense grooming. Such uninterrupted grooming for roughly 25 min is due to presence of the neurotransmitter DA in the venom . This moDrosophila, A. mellifera, Caenorhabditis elegans and others  and D2-like (DOP3) receptors have also been characterized with similar and conserved features in d others . DOP1 red others . With thThe actions of dopamine are mediated via G protein-coupled receptors (GPCRs). Dopamine receptors are members of the rhodopsin-like family of GPCRs, which are characterized by seven trans-membrane spanning domains that contain the ligand-binding site, an extracellular amino-terminus, and an intracellular carboxyl-tail . The jewCharacterization of DAR involvement in post-envenomation grooming relied heavily on use of EEDQ. This agent is a potent dopaminergic neurotoxin that binds irreversibly to vertebrate D1 and D2 receptors, inducing a marked decrease in DAR density in the striatum . ResultsOur pharmacological approach using specific DAR agonists and antagonists in cockroaches reveals the contribution of D1-like receptors in venom-induced grooming. Increased grooming behavior evoked by the specific D1-like agonist SKF38393 was not observed in cockroaches subjected to the D2-like agonist bromocriptine. The same hypothesis was supported by experiments performed in rats and insects, where SKF38393 was shown to stimulate adenylyl cyclase and produce enhanced grooming . BromocrDrosophila brain by activating the D2-like receptor DD2R. (We interpret actions of these agents on walking behavior as follows. After D1 agonist injections, walking behavior significantly decreased. This is not surprising, since grooming increased and both behaviors are mutually exclusive. After D2 agonist injections, walking behavior also significantly decreased with no accompanying increase in grooming. One possible explanation is that overloading the DARs with DA agonist leads to desensitization of post-synaptic DARs. Another non-exclusive alternative is that the DA agonist binds to autoreceptors of the presynaptic membrane causing a decrease in DA release and reduction of walking behavior . Vickreyor DD2R. . Yet, thTo explore further the possibility of D1-like receptor involvement in grooming behavior, specific, reversible antagonists of DARs were injected into the CX prior to EEDQ. This procedure was intended to protect D1-like receptors selectively from EEDQ antagonism, thus allowing for selective removal of D2-like receptors by EEDQ. With this rationale, injection of the reversible D2 selective S-sulpiride prior to EEDQ treatment preserved the D2-like receptors, allowing for selective D1-like antagonism through EEDQ treatment. In contrast, injection of the D1-like receptor antagonist SCH23390 prior to EEDQ preserved the D1-like subfamily only. With these procedures, stung cockroaches injected with S-sulpiride prior to EEDQ did not show normal sting-induced grooming behavior. On the contrary, stung cockroaches injected with SCH23390 prior to EEDQ showed normal sting-induced grooming behavior. Together with the previous results, this suggests a major involvement of D1-like receptors with grooming behavior.Drosophila brain exhibits intense D1-like receptor immunoreactivity in the central complex (The adult  complex . More sp complex . Hence,  complex .According to the results discussed thus far, DAR signaling in stung cockroaches appears to be essential for spontaneous and venom-evoked behavior. After the wasp sting, the escape behavior of cockroaches is also affected. More specifically, during the long term hypokinetic state evoked by the venom, cockroaches, although not paralyzed, fail to escape aversive stimuli . To inveIn conclusion, our findings strongly indicate that D1-like receptors are involved in venom-induced grooming behavior exhibited by stung cockroaches. Furthermore, DAR signaling appears to be necessary for induction of long-term hypokinesia by envenomation.Flies and cockroaches perform distinct bouts of stereotypic grooming movements . Interes


By applying two different imaging methods on this model, we discovered that the transfer through the VR barrier is size-dependent since 40 nm polystyrene particles are more easily taken up in the retina than 100 and 200 nm sized particles. In addition, we found that removing the vitreous, as commonly done for culture of conventional explants, leads to an overestimation of particle uptake, and conclude that the ultimate barrier to overcome for retinal uptake is undoubtedly the inner limiting membrane. Damaging this matrix resulted in a massive increase in particle transfer into the retina. In conclusion, we have developed a highly relevant ex vivo model that maximally mimics the human in vivo physiology which can be applied as a representative test set-up to assess the potential of promising drug delivery carriers to cross the VR interface.Retinal gene delivery via intravitreal injection is hampered by various physiological barriers present in the eye of which the vitreoretinal (VR) interface represents the most serious hurdle. In this study, we present a retinal explant model especially designed to study the role of this interface as a barrier for the penetration of vectors into the retina. In contrast to all existing explant models, the developed model is bovine-derived and more importantly, keeps the vitreous attached to the retina at all times to guarantee an intact VR interface. After  Next to the tragic impact of vision loss on the lives and the surroundings of these people, the health care costs associated with blindness are immense  injection, a technique that is considered safe, minimally invasive and relatively easy to perform  influences their transport over the VR interface. Finally, we apply the presented model to draw conclusions on the drug delivery barrier role of the separate parts of the VR interface.To address this need, we developed an 2.2.1.®) were purchased from Molecular Probes™: 40 nm (8795), 100 nm (F8800), 200 nm (F8809). Dyes for Müller cell and viability staining were obtained from Invitrogen: Hoechst 33342 (H3570), FM® 1-43 (T3163), Mitotracker® Deep Red (M22426), Propidium iodide (P3566). Antibodies against glutamine synthetase (ab73593) and Collagen IV (ab6586) were purchased from Abcam; AlexaFluor® 647 tagged secondary antibody (A27040) was obtained from Invitrogen. Cell culture materials were mostly acquired from Gibco™: CO2 Independent medium (18045088), Neurobasal®-A medium (10888022), Advanced DMEM medium (12491023), B-27® supplement (17504044), Penicillin–streptomycin (15140122), l-Glutamine (25030081), Trypsin–EDTA 0.25% (25200072).Carboxylated polystyrene beads . For this purpose, the FluoSpheres® are diluted a thousand times in HEPES buffer  prior to performing the measurements at 25 °C. Size measurements are done in triplicate with three runs per replicate, and presented based on the number distribution. The zeta potentials are calculated from the electrophoretic mobility of the FluoSpheres based on the Henry equation considering the Smoluchowski approximation. Zeta potential measurements are done in triplicate with two runs per replicate.The hydrodynamic size and zeta potential of the FluoSpheres2.3.2 independent medium until dissection. After removing all extra-ocular connective tissue and disinfecting the eyes by soaking them in 20% ethanol, the sclera is punctured with a 21G needle around 10 mm below the limbus. This hole next serves as an entry point for the scissors used to bisect the eye. After bisection the eye, the vitreous is removed and the posterior eye cup is filled with cold CO2 independent medium. Next, 3–4 flaps are cut in the eyecup, preferably along large veins. While the whole structure is submerged in medium, a trephine blade (Beaver®) with 10 mm diameter is used to isolate a circular piece of retina from each flap. The explant is then removed from the eyecup by gently pipetting medium below. Two of these explants are then placed on a dry 75 mm Transwell® explant filter after which the explant filter is moistened with explant culture medium  and 10 ml of the same medium is added below the explant filter. Finally, the explants are incubated at 37 °C and 5% CO2.Fresh bovine eyes are obtained from the local slaughterhouse where they are enucleated up to 15 min after the animal is sacrificed. The eyes are transported and kept in ice cold CO2.4.2 independent medium at room temperature, a crucial step to allow smooth separation of the retina from the RPE-choroid layer in step E of the protocol. As for conventional explants, the sclera is punctured with a 21G needle around 10 mm below the limbus (step A). In step b, the eye is bisected so that a posterior eye cup filled with vitreous gel remains. Next, at the rim of the eyecup, the retina is gently detached from the choroid using a fine pincet, except at the side of the optic nerve (step C). Then, the vitreous is gently pulled down during which the attached retina slides along (step D–E). After cutting the optic nerve (step f), the whole tissue is slid with vitreous side upwards into a culture dish of 10 cm (Corning) filled with cold CO2 independent medium. In step g, a scalpel is used to cut up to three pieces of VR explant (up to 1.5 cm2) from the vitreous side downwards through the retina. Hereby the potentially damaged edges of the retinal tissue are avoided. In step I–K, a plastic Pasteur pipette with a cut tip is used to gently aspire the VR explant and to transfer it to a dry explant filter from a 75 mm Transwell® dish . Excess amounts of vitreous (e.g. lying next to the retina) can then be removed by aspiration of some vitreous using a plastic Pasteur pipette while cutting through the gel with scissors. Finally, 10 ml of supplemented Neurobasal®-A medium is added below the explant filter (Step L), and the VR explant is placed in an incubator with 5% CO2 at 37 °C.Our newly developed dissection protocol differs from the conventional one by the preservation of vitreous and an intact ILM during dissection. The preparation of this so-called ‘vitreoretinal explant’ is shown in 2.5.15 nanoparticles/ml) were injected (using a 30G needle) in the vitreous layer covering the retina. Multiple injections (about 50 µL per injection) within one VR explant were performed. The injections were done ‘horizontally’ to avoid direct transfer of nanoparticles into the retina  directly or after preparing cryosections (see below). For ‘direct imaging’, a 60× water dipping objective  with a large working distance (2.8 mm) was pushed on top of the vitreous which allowed to image from the vitreous until the ganglion cell layer (Fixed ll layer .2.7.® O.C.T. (Sakura) with liquid nitrogen, 14 µm sections were cut with a cryostat (Leica). For reliability of our results, we applied the workflow drawn in After fixation, the fixative below the explant filter is removed and replaced by 30% sucrose and incubated overnight at 4 °C. After snap freezing the samples in Tissue-Tek2.8.2. Based on these results, the various sections were categorized in three categories i.e. samples containing a low (<10), higher (10–30) or very high (>30) amount of particles per 1000 µm2.To analyze the uptake of the PS beads by the retina, following the workflow presented in 3.3.1.VR explant differs from the conventional explant by the presence of an intact VR interface (e.g. vitreous and ILM). Despite this seemingly small difference, the search for a dissection protocol suitable to obtain the VR explant was intensive. The dissection protocol as presented in ex vivo models developed for IVT drug delivery studies  dye Propidium Iodide (PI) was added to identify cell viability. The cryosections in 3.2.Following the successful characterization of the morphology and viability of the VR explant, we applied the model to study, for the first time, the size-dependent penetration of polystyrene (PS) beads over the intact VR interface of a large animal. The three selected sizes of the PS beads correlate with the sizes of  gene carriers currently under investigation for the treatment of retinal diseases, while 40 nm ranges at the upper size limit of viral vectors, the 100 and 200 nm sized beads represent the size of most common non-viral vectors. ex vivo IVT injections. 24 Hours post-injection, the transfer of the NPs from the vitreous into the retina was investigated using the direct imaging method. Here, a water dipping objective is gently pushed on top of the vitreous to image the vitreous and NFL/GCL layer , low (10–30 NPs/1000 µm2 as observed with 100 nm NPs) and medium (<10 NPs/1000 µm2 for 200 nm NPs). Also on these images, the high VR transfer into the retina of 40 nm beads in comparison with the larger particles is clearly noticeable.To rule out any bias, cryosection preparation was performed complying with the workflow drawn in 3.4.ex vivo model that keeps the entire VR interface intact, we have compared the transport of 100 nm sized PS beads into the retina using respectively our VR explants and conventional explants without vitreous. As can be derived from 2 nearly tripled (11–31%). We furthermore observed that absence of vitreous allowed larger particle aggregates to enter the retina  expression over the inner retinal tissue should be obtained , originally introduced by Russell and Burch as guidelines for more ethical and less use of laboratory animals . Unfortunately, we were unable to confirm a clear co-localisation of NPs and Müller cells using cryosection imaging.Once NPs passed the VR interface, they were generally located in the inner retinal layers, which is in line with the rationale of exploring IVT injection as a valuable strategy for gene therapy, primarily for targeting cell types in the inner retina. Using the direct imaging method we further observed that 40 nm particles co-localized specifically with Müller cell endfeet , an obseInterestingly, while 100 nm sized particles had difficulty crossing the VR interface in our hands, some reports discuss the easy entry of larger particles into the retina (Bourges et al., To illustrate the importance of preserving the VR interface, we have investigated the uptake of 100 nm sized NPs in conventional explants without vitreous. At the same time, this allows us to explore the specific barrier roles of the different parts of the VR interface. Removing the vitreous had a positive effect on retinal uptake, since the fraction of explants containing high amounts of NPs increased and larger NP aggregates were able to enter the inner layers of the retina . Notablyin vitro results, are competent in crossing the different hurdles connected to the VR interface. However, despite its value, some general remarks can be made. It should be noted that by isolating the vitreous along with the retina, the vitreous loses the natural support of the eyecup, causing a partial collapse of the vitreal collagen network (Nickerson et al., in vivo situation, certain dynamic processes present in the living eye are not taken into account such as clearance mechanisms and the anterior-posterior flow present in the vitreous (Xu et al., ex vivo system that most closely resembles the in vivo situation, is the perfused eye model. In this approach, an entire eye is isolated, cannulated and perfused, keeping the eye globe perfectly intact (Mains et al., in vivo studies remain indispensable as for example retinal gene expression usually only reveals itself after 2 weeks (Mowat et al., The significant role of the vitreous and ILM as a barrier for drug delivery and the influence of vitreous removal on the integrity of both of them again highlights the necessity of the model presented in this study. Certainly, our bovine-derived VR explant could be highly valuable as a relevant set-up to assess if promising particles, showing favorable 5.ex vivo models, such as the one presented in this paper, could have great significance in reaching this goal. Surely, our VR explant is currently the most representative ex vivo model on the market that is viable for a sufficiently long time to study carrier uptake. Moreover, our ex vivo approach is readily accessible, relatively cheap and transferrable to other large species like pig or human.For future research, we argue that further detailed characterization of the properties and composition of the vitreous and ILM across species could help to identify which models are the most suited for IVT drug delivery studies with the retina as main target. Thanks to the valuable research performed on especially developed large animal models we have the knowledge to smartly design vectors able of overcoming the vitreous as a barrier. In contrast, owing to the diversity of vectors studied on a variety of species the physicochemical requirements to efficiently cross the ILM is way less coherent. Well-designed systematic studies into which particle properties do result in successful retinal entry from the vitreous could therefore form a sound foundation for future design of ‘the’ optimal gene carrier administered by IVT injection. We are strongly convinced that advanced


The platelet-to-lymphocyte ratio (PLR) and C-reactive protein (CRP) level are markers that have been reported to predict the histological type of various tumors, and here, we evaluated their utility in predicting colorectal polyp histological types.We retrospectively reviewed 172 patients with colorectal polyps who underwent endoscopic polypectomy. The associations between histological type and clinicopathologic parameters were assessed by multivariate analysis.P = 0.002) and CRP (P = 0.009) values were associated with the histological type according to the univariate analysis, whereas low PLR (P ≤ 0.001) and CRP (P = 0.017) values were independent risk factors in the multivariate analysis together with maximum tumor diameter (P ≤ 0.001) and tumor number (P = 0.0014).The optimal PLR and CRP cut-off values were 113.32 and 0.39, respectively. The PLR (Preoperative PLR and CRP are correlated with the colorectal polyp histological type. Previously, the platelet-to-lymphocyte ratio (PLR) and C-reactive protein (CRP), which are two inflammation-related biomarkers, were confirmed to be prognostic indicators in early gastric cancer, gastrointestinal cancers and CRC.However, to our knowledge, no studies have reported the association among PLR, CRP and histological type of colorectal polyps.P < 0.001), number of tumors , a CRP level > 0.39  and a PLR > 113.32  were independent variables for predicting the histological type of colorectal polyps, whereas location and physician were not Table Further multivariate logistic regression analysis revealed that a maximum tumor diameter > 1.35 cm  is one of the most common malignancies of the digestive tract, and its mortality rate in men and women has increased so that it now ranks third in terms of cancer-related deaths among global malignancies . ColonosRegarding the assessment of the histological type of colorectal polyps, the number of cases of colorectal polyps that are diagnosed and treated by colonoscopy has now increased, and the pathological type is subsequently diagnosed by an experienced pathologist. However, considering economic efficiency and feasibility, colonoscopy techniques are difficult to use extensively in the clinic. Therefore, an effective biomarker that can be conveniently implemented for widespread use for the prediction of the histological type in patients with colorectal polyps is urgently needed.Recently, many studies have shown that the systemic inflammatory response is related to tumor progression. The platelet-to-lymphocyte ratio (PLR) and C-reactive protein (CRP), which are two inflammation-related biomarkers, have been confirmed to be prognostic indicators in early gastric cancer, gastrointestinal cancers, and CRC –8. HowevTherefore, this study was designed to assess the relationships among PLR, CRP and colorectal polyp histological type and to identify simple and practical inflammation-related variables and clinicopathological factors that can be used to predict the histological type of colorectal polyps.We retrospectively evaluated the data of 172 patients with colorectal polyps who underwent endoscopic polypectomy and who were diagnosed by postoperative pathologic analysis from July 2018 to September 2019 at Shandong Provincial Hospital affiliated with Shandong First Medical University, China. The exclusion criteria for this study were (1) no pathological diagnosise of the polyp, (2) severe infection, (3) hematopathy, and (4) malignancy. Most patients were excluded due to a lack of complete case data. Eventually, 172 patients with colorectal polyps were included in this study. The clinicopathological data, including sex, age, maximum tumor diameter, location, physician, number of tumors, smoking status, and drinking status, were collected by two reviewers who did not participate in the data analysis.This study was approved by the Ethics Committee of the Shandong Provincial Hospital affiliated with Shandong First Medical University. All patients provided written informed consent.Endoscopic resection (ER), including endoscopic polypectomy (EP), endoscopic mucosal resection (EMR) and endoscopic submucosaldissection (ESD) are used to remove superficial neoplasms from the colon. Endoscopic submucosal dissection (ESD), has been introduced to remove the larger colorectal polyps. EMR and ESD require submucosal injections than conventional snares polypectomy or biopsy techniques. EMR requires the removal of the diseased mucosa with a high frequency trap, and the specimens recovered from the basket are sent for pathological examination. ESD needs to be labeled around the lesion and complete dissection is performed with a scalpel.All patients underwent a preoperative hematology examination. All patients fasted for 6–8 h prior to the blood draw. Blood samples were collected in ethylenediaminetetraacetic acid tubes within 7 days before surgery . Routine blood tests were performed using an automated hematology analyzer  and included neutrophil, lymphocyte, and platelet counts. Liver function tests were performed using a different automated hematology analyzer  and included measurements of the CRP levels.The PLR is calculated as the platelet count divided by the lymphocyte count. Since PLR and CRP have not yet been reported to be associated with the histological type of colorectal polyps, a receiver operating characteristic (ROC) curve was used in this study to determine the most appropriate cut-off values . The valP < 0.05 (two-tailed) was obtained in the univariate analysis. P < 0.05 (two-tailed) was considered statistically significant, and all statistical analyses were performed by SPSS software .Continuous variables are expressed as the mean ± standard deviation and the median (minimum: maximum). The box plot shows the distribution of PLR and CRP in the tubular adenoma group and the tubulovillous adenoma group. The χ2 test was used for the univariate analysis. To determine independent risk factors, a multivariate logistic regression analysis was performed for variables for which Of the 172 patients with colorectal polyps,130 patients (75.6%) were men and 42 (24.4%) were women; their mean age was 55.4 ± 10.5 years. Postoperative pathological analysis confirmed tubular adenoma in 115 patients (66.9%) and tubulovillous adenoma in 57 patients (33.1%). Other clinicopathological characteristics, including sex, age, maximum tumor diameter, location, physician, number of tumors, smoking status, and drinking status, are shown in Table The PLR and CRP values were significantly higher in the tubular adenoma group than in the tubulovillous adenoma group Fig. .Fig. 1DAccording to the ROC curves, the area under the curve (AUC) values of the PLR and CRP were 0.590 : 0.502–0.678) and 0.576 (95% CI: 0.483–0.669), respectively, and the corresponding optimal cut-off values for the PLR and CRP were 113.32 and 0.39, respectively  and CRP , location , maximum tumor diameter , physician  and number of tumors  were significantly related to the histological type of colorectal polyps. Nevertheless, no significant associations were observed between histological type and age, sex, smoking, drinking, neutrophil count, lymphocyte count, platelet count, monocyte count, NLR or the lymphocyte-to-monocyte ratio (LMR) (Table P < 0.001), number of tumors , a CRP level > 0.39  and a PLR > 113.32  were independent variables that could predict the histological type of colorectal polyps, whereas location and physician were not  adenomas, villous adenomas, and severely stunted adenomas are 3, 17, and 37%, respectively . One stuThe link between inflammation and malignancy has been well established since Virchow first proposed it in 1863 . InflammP = 0.022). Lian et al. [P value = 0.000000), and the PLR was thus considered an independent risk factor. However, few articles mention PLR and CRP as predictors of colorectal polyps. A study of 60 patients with colorectal adenomatous polyps showed that serum CRP concentrations were higher in patients with adenomas located proximally (8.674 ± 9.19 μg/ml) compared with the control group  and that CRP levels may be associated with the development of tumors in the proximal part of the colon. To date, no study has reported the relationships among PLR, CRP and the histopathological types of colorectal polyps.Ample evidence supports the prognostic value of PLR and CRP in some solid tumors. Koji et al.  recruiten et al.  retrospen et al.  retrospeIn this study, we first discussed the significance of the PLR and CRP levels in their ability to predict the histopathological type of colorectal polyps. The results showed that in the tubular adenoma group, the platelet counts and CRP levels were higher than those in the tubulovillous adenoma group and the lymphocyte counts were lower than those in the tubulovillous adenoma group, but the differences were not statistically significant. Based on this result, we speculate that the detection of these factors in colorectal polyps may not be sensitive enough to reflect these single differences during the early stages of tumor progression. However, the PLR amplifies the differences in the single distribution. Here, we found a significant difference in the PLR distribution between tubular and tubulovillous adenomas. Since no study has assessed the correlation between the PLR or CRP level and the histopathological type of colorectal polyps, ROC curves were used to determine the cut-off values. In the univariate and multivariate analyses, both low PLR and low CRP were associated with the histopathological types, while low PLR and low CRP were shown to be independent risk factors.Various studies have recommended different cut-off points for the PLR (between 106 and 130) and CRP (approximately 1 mg/ml). In this study, the cut-off point of the PLR was 113.32, while the cut-off point of CRP was 0.39 , 30. VarThis study has several limitations. First, this is a retrospective analysis of patients at a single center. Second, our study was limited to patients whose histopathological types were confirmed as tubular adenoma or tubulovillous adenoma, and we did not analyze patients with other pathological types of colorectal polyps, and thus, our sample size was small. The second limitation was that we assessed only preoperative laboratory values. Further studies are needed to understand the role of the PLR and CRP alone or in combination with other inflammatory biomarkers in predicting the histopathological type of colorectal polyps.This study is first to investigate the relationship between inflammatory factors (PLR and CRP) and histopathological type in patients with colorectal polyps. We found that both the PLR and CRP are novel preoperative biomarkers that are closely related to the histopathological types of colorectal polyps. In our study, the PLR and CRP were shown to be economical, reliable, and convenient tools for predicting the pathological type of colorectal polyps.


M = 74.4; SD = 7.2 years) participated in this study. The initial 18-item version of the scale was constructed, response characteristics examined, and then subjected to factor analysis, as well as evaluation of the scales' reliability and validity. The analysis of response characteristics and subsequent factor analysis resulted in the elimination of eight of the 18 test items. The SWB-HL Total score was derived from the 10 remaining items. It was shown that the SWB-HL tapped three underlying domains interpreted as: Life Satisfaction (three items); Acceptance of Hearing Loss ; and Social Support (three items). Psychometric analysis showed very good reliability and good criterion validity was established for the 10-item SWB-HL Total score. In addition, significant differences were observed between aided and unaided SWB-HL Total scores following 4–6 weeks of hearing aid use. The SWB-HL is a 10-item self-report measure of SWB that shows good reliability and validity when used by older adults with hearing loss and reveals improved SWB following the use of hearing aids.The objective of this research was to develop and evaluate a self-report measure of subjective well-being (SWB) for use with older adults with hearing loss (HL). A convenience sample of 173 local volunteers between the ages of 60 and 88 years ( Recognizing the burgeoning population of older adults worldwide and the importance of facilitating healthy aging, on December 14, 2020, the United Nations adopted a resolution declaring 2021–2030 as the “Decade of Healthy Aging”  when asked first about dating frequency but uncorrelated (r = −0.12) when the order was reversed. When another group of respondents was informed that queries would be made about “two areas of life that may be important to overall well-being,” the responses were again uncorrelated (r = 0.15). This suggests that measures of SWB which incorporate queries about hearing loss will attune the respondent to the importance of hearing loss when responding to queries about their general SWB.Perhaps a measure of SWB can be primed to be sensitive to the impact of hearing loss on SWB. Self-report surveys in general reveal broad effects of question context  organization “…manages the distribution and information regarding administration, scoring and interpretation of a range of questionnaires that measure health-related quality of life for people with chronic illnesses.” At the time of our search, one available measure seemed closest to our needs: Functional Assessment for Non-Life-Threatening Conditions (FANLTC). The FANLTC (Version 4) consisted of a total of 26 items, seven on physical well-being, seven covering social/family well-being, five dealing with emotional well-being, and seven concerned with functional well-being. The first seven items on physical well-being were deleted as they dealt with unlikely consequences of hearing loss including whether the respondent felt ill, had nausea, was bothered by treatment side effects, had pain, and so on. This scale on physical well-being reflects the roots of the FACIT organization's measures which began with the Functional Assessment of Cancer Therapy  were ineligible. Of the remaining 388 eligible individuals, 154 (39.7%) enrolled and purchased hearing aids with 143 completing the measures included in these analyses. They paid the full purchase price for the devices at the time of enrollment. Of the 234 eligible candidates who opted not to purchase hearing aids and enroll, 36 agreed to return to complete several of the pre-fit measures completed by the hearing-aid purchasers with 30 of these individuals completing all the measures included in these analyses. The total sample for these analyses was 173, including 143 individuals who were fitted with hearing aids and 30 who opted not to pursue hearing aids.Of the 173 participants, 102 (59.0%) were male, 65 (37.6%) were female, and 6 (3.5%) did not indicate their gender. Participants ranged in age from 60 to 88 years with a mean age of 74.4 years (SD = 7.2 years). For 16%, formal education ended at high school, whereas 12.1% completed college, another 23.1% held a Masters' degree, and an additional 17.3% held a doctorate or medical degree. Regarding income, total annual income for the preceding tax year was reported to be >$45,000 annually by 55.6% with annual incomes of $5,000–15,000, $15,000–25,000, $25,000–35,000, and $35,000–45,000 reported by 7.6, 11.1, 13.5, and 12.3% of the participants, respectively. When queried regarding the duration of their hearing loss, the median response was 5.5 years with an interquartile range of 3–10 years. Regarding current or prior hearing aid use, 33 (19.1%) were currently using hearing aids and 47 (27.2%) reported ever having worn hearing aids with 14 (8.1%) no longer using them.Informed consent was obtained from all participants and they were paid on a per-session basis for their participation. This study was approved by the Indiana University Bloomington Institutional Review Board.In addition to an audiological exam, several other measures pertinent to this study were obtained. These measures were included, in part, to evaluate the validity of the SWB-HL. The measures pertinent to this study could be divided into two categories: (1) psychological measures of affect or stress/anxiety, personality, and cognition; and (2) measures of communication performance and the participant's reaction to difficulties experienced. For the latter, we relied on the Communication Profile of the Hearing Impaired , as noted in the introduction, is a brief self-report measure of affect and has been used frequently to capture the two affect components of the tripartite SWB model. The respondent is presented with a list of 20 words in a column and, for each, is asked to indicate whether he or she generally feels this way “on a regular basis” and selects among “very slightly or not at all,” “a little,” “moderately,” “quite a bit” or “extremely” as responses, with points assigned from 1 to 5, respectively. Half of the 20 items convey positive affect, such as “excited,” “strong,” “enthusiastic,” “proud,” and “inspired,” whereas the other have convey negative affect, such as “distressed,” “upset,” “scared,” “ashamed,” and “nervous.” Two scores emerge: PANAS-positive and PANAS-negative. Each represents the total points for each set of 10 items, scores ranging from 10 to 50. For the positive scale, higher scores, and, for the negative scale, lower scores, reflect more positive affect.The State-Trait Anxiety Inventory  to match target in the coupler and were then fitted to the patient and verified using real-ear probe-tube microphone measurements with adjustments to better match targets performed as needed. The root-mean-square error between target and measured real-ear gain averaged across frequency (250–6000 Hz) was ≤ 5 dB.One group received four-channel wide-dynamic-range-compression (WDRC) circuits housed in full-concha ITE shells, half with directional microphones and half with omni-directional microphones. The other group received 6-channel open-fit mini-BTE devices with directional microphones. The directional microphones were a fixed super-cardioid configuration, and its function was verified using Verifit software and hardware . Verification of directional performance was obtained in the test box from both hearing aids. The frequency response of the hearing aids programmed for the directional microphone function was equalized to match the frequency response of the hearing aids when set to the omnidirectional-microphone mode. The same basic protocol was used to set and verify target gain for each participant in each group. First, based on audiological information obtained from each participant , target 2-cmThe prescriptive procedure used to generate gain targets was NAL-NL1. With each group and technology, software from NAL was used to generate NAL-NL1 targets, rather than the manufacturer's version of that prescriptive protocol. Within a given group of participants, all were fitted bilaterally with identical make and models of hearing aids. In addition, participants paid the typical clinic price for the devices at the time of delivery and then were paid as research subjects for return visits during which they completed a variety of outcome measures.After on-ear verification of real-ear gain, the participant was counseled about the use, function, and care of the hearing aids. Approximately 4–6 weeks post-fit, the participant returned to complete several outcome measures, with the focus here on the SWB-HL. Reports for several of the conventional outcomes have been published previously for subsets of the study sample reported here with a focus on differences in outcomes for various technologies , the Cohen's d effect sizes  for the 12 items that were retained. In other words, for the 12 remaining items of the SWB-HL, assumptions about the underlying scale did not impact the item scores and parametric tests could therefore be applied with confidence in the validity of those analyses. In all cases, the six items eliminated had skewed distributions with ordered-probit means ≥ 3.7 (maximum = 4) and skewness values exceeding −1.2, consistent with an extremely skewed distribution associated with a ceiling effect. The 12 items that were retained following this analysis of response distributions were items 1, 2, 3, 4, 5, 8, 11, 13, 15, 16, 17, and 18.Liddell and Kruschke  provideds Cohen,  were smaomega function in the R psych package to determine whether all 10 items were represented by a general (g) underlying factor , for the Life Satisfaction, Social Support, and Acceptance of Hearing Loss scales, respectively. This again reinforces that a single 10-item SWB-HL Total score will capture variations in each of these three domains.The SWB-HL could be used to generate four scores based on the general factor, g, and each of the three domain-specific factors. Although the analyses of the SWB-HL support the existence of three domains captured by the 10-item scale, it is not recommended that such individual subscale scores be used due to the small number of items, 3–4, comprising each scale. Instead, it is recommended that just the SWB-HL Total be used with the knowledge that it is tapping the constructs of Life Satisfaction, Acceptance of Hearing Loss, and Social Support. The full scale, 10 items, is itself brief and it is well-known that the reliability of any measure decreases as the number of items comprising the measure decrease. Moreover, the correlations of the SWB-HL Total score with the three subscale scores were ii), and examination of the change in Cronbach's alpha following deletion of each item. The reliability analyses were completed using the alpha function of the R psych package and the polychoric correlation matrix for the 10 items. Cronbach's alpha for the SWB-HL Total score was 0.85  showing strong internal consistency of the measure. In general, the desired range for the mean inter-item correlation is 0.15 < rii < 0.50 . It should be noted that, although several of these scales make use of Likert-type responses, factor analysis does not appear to be impacted by assumptions of ordinal or interval data except for extremely skewed ordinal distributions  personality scale  were z-transformed and added as additional independent variables to each regression equation.p < 0.05) Beta coefficients in the standardized regression equation are highlighted in bold font and the associated zero-order, partial, and part correlations are italicized for those significant contributors. Five significant predictors emerged for the SWB-HL Total score with the solution accounting for 48% of the variance (r2). In addition, collinearity among the predictors was not an issue with the Variance Inflation Factor (VIF) < 1.62 and the Condition Index < 2.7 for all predictors.The linear multiple-regression analysis summarized in Perhaps as interesting are the factors that proved not to be associated with SWB-Total performance. These include the severity of hearing loss, age, and cognition. Associations between cognitive function and general SWB have been observed previously  = 4.29, p < 0.001] with a medium effect size . Significant improvements in SWB were observed following 4–6 weeks of hearing-aid use with the SWB-HL Total.Another form of validation which focuses on the sensitivity of the instrument is to examine the scores before and after intervention  = 2.33, p < 0.05] but with a small effect size . When just the four items tapping Acceptance of Hearing Loss were totaled for evaluation pre- and post-fit, the post-fit mean  was significantly greater  than the pre-fit mean  and a medium effect size was observed (d = 0.37). Given that the hearing aid intervention addresses the hearing loss of the respondent, and consistent with the prior literature on the demonstration of domain-specific improvements in SWB following hearing-aid use, the increased score for SWB-HL items tapping Acceptance of Hearing Loss is expected. More importantly, the increased score for the 6-item SWB-HL score calculated for the general SWB items reflects broader improvements in SWB from amplification in older adults with hearing loss. Again, such subscale scores of the SWB-HL are not being recommended here for use but as a means to demonstrate that hearing aid use impacted more than just the hearing-related items of the SWB-HL.Perhaps the aided improvement in SWB-HL Total score is carried exclusively by the four items of the SWB-HL in the Acceptance of Hearing Loss domain. This pattern would be consistent with the prior literature reviewed in the Introduction suggesting that only domain-specific improvements are expected in SWB measures. Given that four of the 10 items in the SWB-HL Total score make queries about the impact of hearing loss perhaps aided improvements of these four items are sufficient to result in the observed improvement in SWB-HL Total score with amplification. To examine this, a 6-item SWB score was computed from the three Life Satisfaction and the three Social Support items. These non-HL SWB scores were then compared for the pre-fit and post-fit measurements. The mean SWB-HL Total “non-hearing loss” score was 19.45 (SD = 3.17) prior to the hearing-aid fit and 19.93 (SD = 3.28) after 4–6 weeks of hearing aid use. A paired-sample p < 0.001). This correlation, coupled with the increase in mean performance for the group, suggest that the general trend across individuals was for the use of hearing aids for this 4–6-week period to improve the measured SWB. The Pearson-r correlation between pre-fit and post-fit SWB-HL Total scores was 0.66 and statistically significant  SWB-HL-Total score as the dependent measure, the best-fitting regression solution accounted for 50.8% of the variance but only had one significant predictor variable, the z-transformed pre-fit (unaided) SWB-HL Total score. Thus, these analyses with the inclusion of a broad range of potential predictors confirm the relationship between pre-fit and post-fit SWB-HL-Total scores illustrated previously in r = −0.42) and ECHO (r = 0.29). This solution, however, only accounted for 34% of the variance compared to nearly 51% when unaided SWB-HL scores were included. The fact that neither affect or ECHO were significant predictors when the unaided SWB-HL total scores were included implies that the unaided SWB-HL Total score captures aspects of the individual's self-reported affect and hearing-aid expectations.If the unaided SWB-HL Total score is not included among the independent measures and the regression analysis predicting aided SWB-HL Total score is repeated, only two significant predictors emerged: affect . In addition, we noted the importance of item context in the Introduction, including the potential impact of priming. The final 10 items comprising the SWB-HL were extracted from the initial larger set of 18 items. It is unknown how the eight items that have been eliminated from the original corpus of 18 may have impacted the responses for the 10 items that remained. Further research is needed to overcome these limitations. In addition, these context effects should be kept in mind by developers of future questionnaires or surveys in the field.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Indiana University Bloomington IRB. The patients/participants provided their written informed consent to participate in this study.LH performed all the data analyses described here and wrote the manuscript without assistance.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Spinal perineural Tarlov’s cysts (TCs) are considered incidental findings that occasionally might exert pressure upon nerve roots and correspond with patients’ signs and symptoms. Purpose of this meta-analysis is to deliver global incidence and characteristics  of TCs.Following PRISMA checklist, all major databases were searched by two authors for radiologic studies reporting incidence and morphologic features  of TCs. Anatomical Quality Assessment tool was applied for risk of bias evaluation. Meta-analysis of random-effects model was employed. Subgroup analysis for regional distribution, gender, sacral levels, age, correspondence with symptoms, and persistent genital arousal disorder (PGAD) were planned ahead.p < 0.001, test of homogeneity, χ2). Subjects with PGAD had incidence of 37.87% (95% CI 2.45–81.75). TCs in pediatric population are rare—0.53% (95% CI 0.02–1.51). 15.59% of TCs corresponded with symptoms.22 radiologic studies of level 3 evidence involving 13,266 subjects were included. Global pooled prevalence of TCs was 4.18% (95% CI 2.47–6.30). Mean pooled sagittal diameter was 11.86 mm (95% CI 10.78–12.93). Sacral cysts strongly prevailed over the other segments. Of the sacral, S2 level was the most common (46.7% [95% CI 29.4–60.5]). Geographically, the highest incidence was found in Europe (6.07% [95% CI 1.49–13.00]), followed by North America (3.82% [95% CI 0.49–9.44]), and Asia (3.33% [95% CI 1.52–5.75]). TCs were more common in women than in men  cysts are found in a minority of population. S2 level of the sacral bone is affected most frequently. There is female predominance. Correspondence with symptoms is seen in less than one-fifth of TCs. Studies with stronger evidence level are needed to corroborate the results. The purported high incidence in PGAD requires confirmation in case–control studies for the risk-ratio calculation.The online version of this article (10.1007/s00276-020-02644-y) contains supplementary material, which is available to authorized users. Spinal perineural Tarlov’s cysts are thought to be common radiological findings, often incidental and asymptomatic. They are a dilation of the nerve-root sheath and tend to communicate with subarachnoid space through a valve-like mechanism, which thus contain cerebrospinal fluid (CSF) as well as neural tissue , 6 Fig.. TraditiMajor electronic databases  were searched by two researchers  using a MeSH term ‘Tarlov cyst’ and corresponding synonyms. Full strategy is presented in Electronic Supplementary Material 1. The search started on the 1st August 2020 and was finalized by 6th August 2020. Year of publication was not restricted. Mendeley Desktop 1.19.4 was utilized for title importation, de-duplication, and creation of bibliographic details. Additionally, references of the included papers were checked for pertinent articles. PRISMA checklist is available as Electronic Supplementary Material 2.Analysis was limited to those studies that met our inclusion criteria: (1) clear incidence of Tarlov’s cysts among a specified group of subjects; (2) the incidence regarded the group of at least 10 subjects. Papers were excluded if any criterion of the following was found: (1) conference abstracts, (2) commentaries, (3) letters to the Editors, (4) insufficient data, (5) incidence of Tarlov’s cysts was aggregated with other lesions and could not be disaggregated, (6) case reports, and (7) series with less than 10 subjects among whom presence of the cysts was evaluated. TK and WO are fluent in English and Polish. In case of other language, professional translators were inquired.Data were extracted by two of us (TK and WO). The following characteristics were of interest: (1) total number of subjects evaluated for presence of Tarlov’s cysts, (2) number of individuals with at least one TC, (3) number of individuals with multiple TCs, (4) total number of TCs in the group, (5) number of patients with only one, only two, only three (and so on) TCs, respectively, (6) number of TCs in sacral, lumbar, thoracic, or cervical region, respectively, (7) number of TCs corresponding with symptoms (excluding back pain) or signs, (8) number of TCs not corresponding with symptoms or signs, (9) number of TCs in patients with persistent genital arousal disorder, (10) number of TCs in patients with transitional lumbosacral vertebra, (11) mean age of the group with TCs, (12) number of adults and children with TCs, (13) size of TCs in millimeters , (14) prevalence of TCs with respect to nerve-root level, (15) prevalence with regard to sex, (16) shape of TCs, (17) region of origin, and (18) modality of imaging. Data were collected only if it could have been disaggregated. In several cases, the disaggregated data were provided by the corresponding authors who were communicated with.Two researchers are TK and WO. Risk of bias was assessed in two ways: quantitatively and qualitatively. Qualitative analysis was performed by means of AQUA tool   independI2 and χ2 were incorporated into evaluation of heterogeneity. I2 value was interpreted with classic approach: 0–40%: not be important, 30–60% moderate heterogeneity, 50–90%: substantial heterogeneity, and 75–100%: considerable heterogeneity. Arbitrary p value of < 10% was set for Cochrane Q significance (< 0.10). Conversely, significance of comparative tests was agreed to standard < 5% (< 0.05). Random-effects model for meta-analysis of pooled prevalence was applied. Subgroup differences in prevalence were compared using test of homogeneity based on χ2 .Formal statistical estimation of pooled prevalence was executed using software MetaXL 5.3, EpiGear International Pty Ltd. . Estimation of mean sagittal diameter was executed with Comprehensive Meta-Analysis V3 . For single-categorical variables, pooled prevalence was estimated with corresponding confidence intervals of 95%. Subgroup analysis was planned with regard to geographical regions, specific entities such as persistent genital arousal disorder, sex, and age (children and adults). Distribution within the sacral spine was also analyzed to determine which sacral level is affected most commonly.Initial search yielded 4762 records including 2 from references of the pertinent articles. Following the de-duplication, 3518 titles and abstracts were checked for relevance to the topic. 41 full-text articles were assessed for eligibility . Thus, mean age of those in the main cohort was 48.9 years. Geographical distribution was as follows: 8 studies from North America (n = 1298), 6 from Europe (n = 5903), 7 from Asia (n = 5808), 1 from Africa (n = 217), and neither from Australia nor South America.Characteristics are presented in Table Q = 461.49, p < 0.01, I2 = 96.1%; see Fig. Q = 269.62, p < 0.001, I2 = 98.15%), followed by North America , Asia , and Africa . Comparing all four regions, the geographical differences were not significant . However, direct comparison revealed that incidence in Europe was significantly higher than in Asia (p < 0.01) or in North America (p < 0.01). Incidence in Asia was not dissimilar from that in North America (p = 0.607). Subjects with persistent genital arousal disorder showed prevalence of 37.87% . Size of the cysts was measured in three studies (n = 1960). Mean pooled diameter was 11.86 mm . Incidence in women was 7.01% , whereas in men 4.05% . Tarlov’s cysts in pediatrics were rare . Shape was oval/rounded in 58.3% of TCs and tubular in 41.7% . Of the cysts that were found, 57.4% were single, whereas in 42.6%, multiple TCs were seen . Only 15.59% of TCs corresponded with symptoms of neural compromise  . The highest general incidence—17.65%—was suggested by Larsen et al. in 1980 . As contp = 0.0003, test of homogeneity, χ2). Literature has not explored the causes of this sex-related predominance, yet. Our hypothesis is that formation of TCs in adult females might be associated with the level of primary female sex hormones, since in pediatrics, when these hormones are usually low, TCs are an exceptional finding [8 studies addressed gender-specific prevalence. In women, TCs occurred at 7.01% (95% CI 2.57–13.21) whereas men had prevalence of 4.05% (95% CI 1.07–8.57). Apparently, females are more often affected by TCs .Sacral perineural cysts were the ones to be documented most consistently. It was a subject of controversy which sacral level is usually involved. Incidence of S2 TCs was declared highest in three studies , 15. S3 Subgroup analysis reveals that patients with PGAD, mostly women, might be more prone to developing TCs. Two of three studies involving those patients gave an account of high TC incidence (50–66.67%) , 17. HowDespite being comprehensive, this meta-analysis is not free from limitations. High methodological bias found in many papers is highlighted, primarily due to the fact that in many of them, measures were not taken to reduce intra-/interobserver variability. Small number of studies about PGAD is the reason of the wide confidence interval of incidence in this group. It, however, indicates that PGAD might be associated with TCs. Therefore, case–control studies would be of value for calculation of the risk ratio. Based on the findings of this study, it is suggested that more studies on incidence of TCs be conducted in South America, Africa, and Australia. Moreover, results of this meta-analysis and the emphasized gaps in knowledge can be used in future research on Tarlov’s cysts. As all but one of the studies assessed only lumbosacral spine MRI and not whole-spine MRI, there exists some risk of bias regarding counting the vertebrae in individuals with transitional abnormalities. Since only a handful of papers delineated morphological features of the cysts such as size or shape, as for now, it is difficult to seek link between these characteristics and clinical picture. Especially, it is still problematic to determine quantitative relation between TCs and the most frequent complaints expressed by patients, which is radicular pain and local back pain. Moreover, there are not enough data to univocally answer the question whether spinal cord or cauda equina might herniate into the TCs. Hence, more precise depiction is encouraged in upcoming studies.Spinal perineural Tarlov’s cysts are found in a minority of population. Women tend to develop TCs more often than men. Perineural cysts in pediatrics seem to be rare findings. Incidence in Europe appears to be higher than in other continents. S2 level of the sacral bone is affected most frequently. Possibly high incidence in PGAD requires confirmation in case–control studies for the risk-ratio calculation. Studies with evidence stronger than level 3 are needed to corroborate the results of this meta-analysis.