Baseline PET tau and amyloid-β predicted decline in episodic memory (p ≤ 0.0001), but isolated PET amyloid-β did not. Isolated PET Tau positivity was only observed in 2 participants (0.71% of the sample). While results for amyloid-β were similar using CSF or imaging, CSF and imaging results for tau and neurodegeneration were not interchangeable. PET tau positivity was superior to CSF p-Tau181 and PET amyloid-β in predicting cognitive decline in the AD continuum within 3 years of follow-up.For early detection of Alzheimer’s disease, it is important to find biomarkers with predictive value for disease progression and clinical manifestations, such as cognitive decline. Individuals can now be profiled based on their biomarker status for Aβ42 (A) or tau (T) deposition and neurodegeneration (N). The aim of this study was to compare the cerebrospinal fluid (CSF) and imaging (PET/MR) biomarkers in each ATN category and to assess their ability to predict longitudinal cognitive decline. A subset of 282 patients, who had had at the same time PET investigations with amyloid-β and tau tracers, CSF sampling, and structural MRI (18% within 13 months), was selected from the ADNI dataset. The participants were grouped by clinical diagnosis at that time: cognitively normal, subjective memory concern, early or late mild cognitive impairment, or AD. Agreement between CSF (amyloid-β-1-42(A), phosphorylated-Tau181(T), total-Tau(N)), and imaging (amyloid-β PET (florbetaben and florbetapir)(A), tau PET (flortaucipir)(T), hippocampal volume (MRI)(N)) positivity in ATN was assessed with Cohen’s Kappa. Linear mixed-effects models were used to predict decline in the episodic memory. There was moderate agreement between PET and CSF for A biomarkers (Kappa = 0.39–0.71), while only fair agreement for T biomarkers  and discordance for N biomarkers across all groups (Kappa ≤ 0.14) was found. Baseline PET tau predicted longitudinal decline in episodic memory irrespective of CSF p-Tau181 positivity ( The rapid development of several biomarkers for Alzheimer’s disease (AD) during recent years has allowed in vivo tracking of the pathological components of the disease. Some of these biomarkers were designed to target AD-specific changes, such as the deposition of amyloid-β (Aβ) and tau, while others target downstream neurodegeneration. Aβ- and tau-specific positron emission tomography (PET) ligands allow in vivo evaluation of the Aβ and tau components of brain pathology, respectively, and the same is claimed to be possible for the fluid biomarkers such as cerebrospinal fluid (CSF) levels of Aβ42 and tau phosphorylated at Thr181 (pTau). A series of biomarkers have also been proposed for downstream neurodegeneration; these include brain atrophy measured by magnetic resonance imaging (MRI), total tau levels in the CSF (tTau), and glucose metabolism changes as assessed by PET . A recenIn this study, we characterized participants in the Alzheimer’s Disease Neuroimaging Initiative (ADNI) according to their cognition status, ranging from normal to dementia, compared to their ATN component status (positive/negative biomarkers). Our aim was twofold. Firstly, we wanted to assess the level of agreement/concordance between the imaging and CSF biomarkers across the ATN components. Secondly, we aimed to evaluate which of the investigated biomarkers best predicted subsequent cognitive decline in episodic memory function, the cognitive domain that is typically affected at the earliest stages of AD.http://adni.loni.usc.edu) on 09/04/2021. Additional information is found at www.adni-info.org. ADNI was launched in 2003 for testing whether neuroimaging biomarkers in combination with clinical, biological, and neuropsychological markers could measure progression in AD spectrum.The data were downloaded from the ADNI website  assessment were initially selected (n = 764). However, only participants who had had CSF samples taken for Aβ42, tTau, and pTau assessment, had had PET Aβ 18F-florbetapir (FBP) or 18F-florbetaben (FBB) assessment on the same visit code as the baseline PET tau, and had also had a structural T1 MRI scan within 13 months of baseline were finally included (n = 282). For 51/282 (18%) subjects, the MRI scans were obtained later than the baseline visit .ADNI participants who had at least one PET n = 90), Subjective memory concern , Early/Late MCI  and AD dementia (n = 24).The subjects were grouped according to their clinical diagnosis at baseline into the following groups: Cognitively normal  were classified as SMC if they had a cognitive change index score (first 12 questionnaire items) ≥16 according to ADNI2 guidelines .The distinction between EMCI and LMCI was based on Clinical dementia rating, Mini-Mental State Examination and education-adjusted severity of episodic memory impairment , following ADNI2 thresholds .The files containing the regional PET uptake data for Aβ  and tau (FTP) were downloaded from the ADNI website (UCBERKELEYAV45_/UCBERKELEYFBB_/ UCBERKELEYAV1451_01_14_2021.csv). These data had been pre-processed using the ADNI pipeline . PET Aβ http://www.adni-info.org/). The concentrations of Aβ42, pTau, and tTau in CSF were obtained from the ADNI depository ; these were measured using Elecsys immunoassays on a cobase 601 analyzer, as previously described [Lumbar punctures were carried out to obtain CSF samples as described in the ADNI procedures manual  have been described previously [Pre-processed data concerning volumetric and cortical thickness measures were also downloaded from the ADNI website (UCSFFSX6_02_05_20.csv). The primary MRI measure was the FreeSurfer (version 6.0)-derived hippocampal volume (HV) after adjusting (HVa) for intracranial volume. The measure is a proxy for neurodegeneration that correlates well with learning and memory cross-sectionally and longitudinally . The caleviously . We alsoeviously .The ADNI (episodic) composite memory score has been validated as a reliable metric for cognitive change in the ADNI cohort . The scoThe concordance of the biomarkers across the ATN framework was evaluated in a descriptive manner, including use of the Cohen’s kappa coefficient. Cohen’s kappa statistics measure inter-rater agreement for categorical items. Cohen’s kappa coefficients are reported with 95% confidence intervals and values >0.40 indicate a relevant level of agreement . The prep < 0.05, not adjusted for multiple comparisons).The correlations between the different biomarkers were assessed with the use of the Spearman’s rho correlation coefficient (Rho) . The biomarker profiles were modeled as factors in two sets of models for assessing: (1) the effect of concordance/discordance of CSF and PET within the same biomarker class (amyloid or tau); and (2) the effect of concordance/discordance of the two biomarkers class (A/T) within CSF or PET. Additional details on the models are reported in the Supplementary Information (SI). Ten models with different combinations of variables were tested and the model with the lowest Bayesian Information Criterion (BIC) was chosen. Tests of significance using LMMs were performed with P ≤ 0.05. In the longitudinal analyses, we evaluated if a particular group had difference in slope (faster decline) in the longitudinal memory follow-up compared to the reference group (-/-). All statistical analyses were performed using R software/environment (version 4.0.2) [Linear mixed-effects models (LMMs) were built for assessing the effect of biomarker status in predicting prospective cognitive decline. The longitudinal ADNI (episodic) memory composite score (ADNI_MEM) was used as the outcome variable; the ADNI-MEM follow-up datapoints were predominantly distributed around 12 and 24 months and to assure that the follow-up across the subjects was homogeneous, and to exclude outliers, in our sample we limited the follow-up interval to 36 months (only 12/527 observations available were excluded), only subjects with at least two ADNI_MEM measures were included  in the CSF panel than in the imaging panel. More specifically, CSF A– cases can be either T+ or T-, while PET T+ cases are usually A+ as also evidenced in Fig. p = 0.01); while in the SMC group, pTau correlated significantly but modestly with tau PET across the ROIs . As for the CI groups, while the EMCI group showed no significant correlation between pTau and tau PET, LMCI and AD demonstrated moderate to strong significant associations across the PET tau ROIs . Furthermore, in the EMCI group, CSF T+ and PET T+  were 24% and 8%, respectively while, in LMCI, 35 and 23%. This indicates that EMCI and LMCI are two distinct groups with regard to tau pathology. Levels of the neurodegeneration biomarkers in CSF (tTau) and imaging (HVa) did not correlate significantly except in the LMCI group . The lack of associations, especially in the CU groups, demonstrates that tTau and HVa levels are not interchangeable to estimate neurodegeneration. Correlations between additional pairs of biomarkers were also tested in the CU/CI and diagnostic groups  of the CSF and imaging biomarkers with regard to the ATN status across diagnostic groups is shown in Fig. For the Aβ biomarkers (panel A), the agreement was moderate across the diagnostic groups, increasing gradually with increasing cognitive impairment. The LMCI group had the highest concordance, while AD group was affected by the greatest statistical uncertainty due to the lack of PET+/CSF- cases.In contrast to Aβ, the Cohen’s kappa for Tau status varied across the diagnostic groups. For discordance, isolated CSF pTau positivity was more prevalent than isolated imaging tau positivity. In agreement with these findings, more supporting results on different Tau ROIs and their comparison can be found in Supplementary Fig. 2a and b.For the neurodegenerative biomarkers, there was no clear pattern of agreement among the diagnostic groups with the exception of the SMC group, where a clear lack of agreement was detected. Concordances between the other combinations of neurodegeneration biomarkers are shown in Supplementary Fig. The ATN profiles resulting from CSF and imaging biomarker results were substantially discordant see Fig.  and 4a,bn = 213 with at least two ADNI_MEM measures). Separate models were applied for the Aβ and tau biomarkers, and ten models were tested (more details in Supplementary Table p = 0.03), but after a visual inspection of the residuals the assumption was considered not violated. With regard to the Aβ biomarkers, only the concordant positive profile (CSF+/PET+) showed a significant negative interaction effect on episodic memory decline , while the estimates of the interaction with time for the other profiles were not significant  in predicting prospective cognitive decline, as measured with the composite ADNI_MEM score , while the estimate of the interaction with time for the CSF+/PET- profile was not significant  showed a significant negative interaction effect with time (relative to CSF-/PET-), suggesting an overall decline in episodic memory in such groups , irrespective of tau status, while the effects of tau positivity and Aβ negativity (A-T+) did not reach statistical significance. The A-T- profile had a generally positive effect on episodic memory, probably as a result of the learning effect of repeated testing , while the A+T- profile did not show this effect  had a negative effect on longitudinal episodic memory performance cognition relative to the control profile A-T-  The CSF and imaging biomarkers resulted in differential ATN profiles, mainly as a result of discordance in the biomarker modalities between the tau and neurodegeneration components; (b) PET tau positivity was a better predictor of short-term cognitive decline than PET Aβ or CSF pTau results.In this study, we started with the diagnosis  and then profiled the pathological burden of the participants from CSF and imaging biomarkers separately, using the ATN profiling system. This approach was used to evaluate concordance between the CSF and imaging biomarkers impartially for the different diagnostic groups and to improve understanding of which biomarker, especially where there was discordance, might provide better accuracy for predicting cognitive decline.We investigated the associations and concordance between ATN biomarkers, with special attention to comparisons of pairs within the same ATN category: CSF Aβ42 vs PET Aβ, CSF pTau vs PET tau and CSF tTau vs MRI-derived neurodegeneration biomarkers. We found that Aβ results correlated more closely between CSF and PET than other biomarker pairs, and agreed across the different diagnostic groups, in line with other reports . InteresWe found relative disagreement between CSF pTau and PET tau results, and even more disagreement between tTau and MRI-based neurodegeneration biomarkers, which indicates the lack of interchangeability between CSF and imaging modalities for monitoring tau accumulation and neurodegeneration. The higher range of values for CSF pTau and the higher proportion of participants across diagnostic groups with T+, relative to PET, points to earlier changes in CSF tau measures in AD spectrum, as previously suggested . HoweverThe studied neurodegenerative biomarkers, more than other pairs of considered biomarkers, seemed to not be interchangeable. This lack of agreement was present across all diagnostic groups and also within the different MRI measures , with no clear agreement pattern across the different diagnostic groups. Even if CSF total Tau is not the best neurodegeneration biomarkers, compared to plasma NfL (for example) , CSF tTaThe ATN profiling confirmed the lack of interchangeability observed in the other analyses between CSF and imaging modalities; the discordance between the two should be taken into account in future guidelines for the in vivo characterization of AD-related pathological changes using biomarkers, instead of the existing algorithm . This isWhen looking at the pairs of neuropathological markers (Aβ and tau together) within each modality for defining the biomarkers most predictive of cognitive decline, different results were seen for CSF and imaging biomarkers. For CSF, Aβ positivity was related to worse cognition longitudinally, irrespective of tau status, although the estimates for this effect were small. For imaging, rapid cognitive decline was predicted by PET tau positivity rather than PET Aβ positivity, since tau positivity was coupled with Aβ positivity but not vice versa. It is known that Aβ positivity per se does not predict short-term cognitive decline, given the age-specific positivity rate of >50% for people aged 80–90 years  and giveAnother issue to consider is the selection of which tau region to use for assessing cognitive decline, since some brain regions  are more affected in the early stages of the disease and might progress slower than others; while others are more affected at the AD stage  . Our datThe follow-up was relatively short (up to 3 years). Despite the large cohort of subjects to choose from, we limited the patient selection to those for whom we had results for all six ATN biomarkers, which limited interpretation of the results and requires validation in other samples. We focused on the dichotomization  and did not investigate the biomarkers as continuous variables. The choice of total Tau as fluid biomarker for neurodegeneration was forced due to the availability of the ADNI data, a more suitable biomarker such as plasma NfL would have been preferrable but was not available for ADNI3 cohort.From these data, it can be concluded that CSF and imaging biomarkers differ considerably within the ATN framework; the most effective of the investigated biomarkers for predicting cognitive decline was the PET tau biomarker, alone. Our results provide support for the prioritization of PET tau over other biomarkers in the assessment of patients with cognitive impairment; if the result is then positive, there is a high chance of rapid cognitive decline. In the presence of a PET tau-positive result, Aβ testing might not be necessary since it is likely that PET or CSF Aβ would be positive too, while CSF tau would show low specificity for detecting short term AD-related cognitive decline. If PET tau is negative, it is suggested that there will not be cognitive decline in the short term (up to 3 years of follow-up time) and other investigations with long-term biomarkers could then be considered.Supplemental Material


Surgical management of hair loss has become an increasingly challenging procedure, when dealing with scar alopecia. We investigated the efficacy of hair transplantation in patients with head and neck scar alopecia.From 2016 to 2018 in Shiraz, Iran, all patients with scar alopecia in head and neck were evaluated for efficacy of follicular unit extraction (FUE), follicular unit transplantation (FUT) or a combination of two methods from donor sites in scalp and beard various hair-grafts were compared. Fifty-six patients were enrolled. Most of them were between 31 and 40 yr old (48.3%) and male (71.4%). Trauma, burn, surgical excision of adjacent skin, radiotherapy and leishmaniasis were the registered causes. Scars were visible in scalp (39.3%), beard (28.6%), eyebrow (21.4%), and moustache (10.7%) regions. FUE (87.5%), FUT (10.7%) and a combination (1.8%) were the used methods. One-hair-grafts were used in eyebrows (100%), moustache (100%), beard (88%) and scalp (7.9%), while 2-hair-grafts in beard (6%) and scalp (47.4%) and 3-haired grafts in beard (6%) and scalp (44.7%) transplantations. In head and neck scar alopecia, hair transplantation was selected based on type and depth of scar. FUE was targeted when huge grafts were not needed, including beard, moustache, and eyebrow, while FIT was used when extensive scars were present in scalp. One-hair-grafts were mostly applied for eyebrow, moustache and beard, 2-hair- and 3-hair- grafts for beard and scalp transplantation. These findings can be added to the literature when FUE, FIT, or their combination are targeted in hair restoration of scar alopecia in head and neck.  Hair loss can happen temporary or permanent and can be local or diffuse due to physiologic, as noted in normal aging, or iatrogenic as observed in burns, trauma, chronic diseases, iron deficiency, thyroid disorders, high fever, consuming some medications, chemo- and radiotherapy, exposure to chemicals, hormonal changes, androgenetic factors, and drastic diets2. Hair loss can occur due to generalized or local skin diseases, such as Lichen planopilaris, lupus, and alopecia areata2. Stress was shown to accelerate a genetically programmed hair loss3.Hair loss is still a significant problem worldwide resulted into enrolling more than 600,000 surgical hair restoration procedures in 20164. As a result, surgical management of scarring alopecia has become an increasingly popular and satisfying procedure for both men and women and this is a more challenge when treating patients with burn scarring alopecia5. Hair transplantation has been conducted for decades and has experienced a cosmetic revolution in the 21st century and identical to all surgical procedures, there has been a continual evolution in this procedure with the goal of an ever safer, efficient, and even higher level of patient satisfaction1. In the earliest hair transplants, a punch was used to extract donor hair follicles for repair of regions of the moustache, beard, and eyebrows scarred by burns6, but recently, surgical management of hair loss has become an increasingly satisfying and popular method7. Scarring alopecia, named as cicatricial alopecia is an irreversible inflammatory follicular damage associated with hair loss7. Consultation is the key point for a successful procedure considering the key points in every consultation including the history of hair loss, the patient’s physical examination, the patient’s goals, the use of medications in treatment of the hair loss, and where to transplant. The key points in the patient’s physical examination are the donor density (hair follicles per cm2), the caliber of the patient’s hair, and the extent of hair loss8. A successful hair transplant procedure should consider the extent of hair loss, density of donor hair, and caliber and color of hair, and also a realistic expectation of the patient and physician. A challenge for the hair transplant surgeon is to reach an acceptable distribution of transplanted hair and creates a permanent natural hairline and over time, the transplanted hair remains cosmetically satisfying for the patient and physician9. FUE was described as a method of harvesting follicular units from a donor site and the surgeon extracts individual follicular units with circular punches (0.8 to 1 mm in diameter) that heal by second intention within a few days10. The harvesting of donor hair follicles can be accomplished by amputating a strip of hair-baring skin called strip harvesting or follicular unit transplantation (FUT) method and by removing each follicle individually via follicular unit extraction (FUE)11. FUE is a procedure that allows to pick desirable follicular units individually without any need to suturing and with minimal scarring12. The procedure involves making a submillimeter punch incision surrounding a single follicular unit extending into the dermis using a motorized sharp punch of 0.9 or 1.0 mm in diameter10. The follicles are then extracted individually by a pair of fine forceps, leaving an indiscernible donor scar10. It is a procedure that makes transplantation surgery less invasive with no need to sutures, with no linear scar and results in a very high percentage of transected follicles. It is a very slow technique as the follicular grafts are extracted individually from various body sources such as scalp and beard13, but the main disadvantage of FUT is formation of a sizable donor scar that can be an issue for patients who prefer shorter hairstyles10. In FUT, surgical removal of a strip of hair-bearing scalp tissue is usually 0.6-1.0 cm wide and 8-20 cm long. Traditionally the donor hair is divided into grafts containing 10-25 hairs each14. In a majority of patients, the donor hairs harvested by FUE technique avoids a linear donor site incision and patients typically can cut the hair as short as desired. FUT is another harvesting technique via the excision of a small elliptical strip of skin, usually from the occipital scalp, also called strip harvesting15. As hair loss due to scarring can have a great impact on the self-esteem and quality of life of patients than the scars themselves and act as a constant reminder of the causative traumatic incident to the patient, the efficacy of FUE, FUT or a combination of two methods from donor sites in scalp and beard was assessed in head and neck scarring alopecia.Burn injuries, whether caused by flame, scalds, chemicals and even electrical injury can affect a large body surface area and can be isolated to the head and neck region leaving severe scarring which is hard to hide with clothing, specifically the scalp, beard region, eyebrows, and eyelashes that makes hair transplantation more challengingFrom Mar 2016 to Mar 2018, all patients from both sexes and various age groups who referred to Novin Iran Clinic in Shiraz, Iran and suffered from scarring alopecia in scalp, eyebrows, moustache and beard due to burn, surgical excision of adjacent skin, trauma, radiotherapy and leishmaniasis were enrolled. Those with a history of skin diseases in active phase such as alopecia areata, Lichen planopilaris were excluded from the study. Treatment plans were made after a thorough history taking of any relevant medical condition or medication, patient-doctor consultation results, physical examination and type and depth of scars. Once a patient scheduled the procedure, preoperative and postoperative guidelines were shared with the patient and a written signed consent letter was provided. An ethical approval was also received from institute Ethics Committee based on Declaration of Helsinki. All patients could contact us with any questions regarding the procedure protocol. During consultation, the type and depth of scars and the proper caliber and density of the hair in the donor region were evaluated. Before treatment, we had an appointment with the patient in front of a mirror with a grease pencil or marker to draw in the area of planned hair placement to reach an agreement on a realistic place for hair placement and if FUT was needed, the optimal donor region was defined.During the procedure, the patient was asked to be in the prone or supine position to allow us a better visibility and stability for donor harvesting and to reduce the incidence of vasovagal episodes during donor harvesting. The patient was premedicated to help relaxation and bearing the hours of surgery much more easily with an oral anxiolytic medicine such as diazepam (10-20 mg), 30 min before the surgery. Patients underwent hair restoration using FUE, FUT or a combination of two methods from donor sites in the scalp and beard based on the type and depth of scars. 16. During the procedure, patients were comfortable and could listen to music, watch a movie, or relax. They could inform us if they were feeling any discomfort to administer more local anesthetics. Patients were instructed to expect a probable swelling along the forehead and eyes for 3 to 5 d after starting the procedure. They were visited in the clinic on the 1st postoperative day to learn how to wash the hair and to take care of the grafts without allowing the shower water to directly hit the grafts until one week. The crusts were gently removed after a week.For all patients if FUT was necessary, the protocol was explained to be an elliptical donor strip provided from posterior scalp. For FUE technique, transplanted hair avoided a linear donor site incision and patients typically could cut the hair as short as desired. The transplanted hair would begin to grow 4 months after the procedure and improve gradually until 12 monthsIn FUE procedure, a submillimeter punch incision was created surrounding a single follicular unit extended into the dermis. Technique and operator standardization were carried out using a specific protocol. Local anesthesia was conducted for all patients using 1% lidocaine together with 1:100,000 epinephrine. Two registered nurses trained by the first author were responsible for FUE and each patient was randomly enrolled by one of the nurses. The punches varied in size ranging from 0.75 to 1.2 mm in diameter attached to a motorized sharp punch . The follicles were extracted individually by a pair of fine forceps. The average procedure in each session took 6 hours. The donor areas were the occiput or the parietal scalp or the beard. Regarding restoration of beard, moustache and eyebrow, all patients received the grafts via FUE technique. One-hair-grafts were mostly applied for the eyebrow, moustache, and beard; whereas 2-hair-grafts for beard and scalp and 3-hair-grafts just for the scalp and beard. When the hairs within a graft were divergent in their growth, 2-hair grafts were not used. After the procedure, patients returned home with a pressure dressing that remained on overnight. They received prednisone (30 mg/d for 3 d) to minimize frontal edema and acetaminophen (2-4 tablets) for pain relief. Patients applied repair creams to the donor site twice daily for 1 week after surgery. Topical antibiotics were administered to the donor area twice daily for a week. They could take shower daily to allow the water to rinse off the scalp, but they avoided scratching or scrubbing at the scalp. The perifollicular crusting around the grafts was removed after one week and shaving was allowed after 2 weeks. Patients could have their make up in the eyebrow area one week post-operation after all the crusts were fallen out.The FUT procedure was done under local anesthesia using 1% lidocaine together with 1:100,000 epinephrine to minimize the bleeding. When the incisions were made, 10 to 30 mL of saline was administered to produce turgor in the donor ellipse and if patients were anxious about pain, they underwent monitored sedation. The procedure was divided into 4 steps of (i) Extraction of the donor strip, (ii) Microscopic dissection of the donor strip into follicular units, (iii) Creation of recipient incisions and (iv) Implantation of follicular grafts. In FUT, the region between the occipital protuberance and 2 cm above the top of the ears was the selected region for harvesting the hair as a band of about 1.5-2.5 cm wide and 30 cm long using a scalpel blade of number 10. The incision was undertaken clean parallel to the line of the hair follicles and at a depth of 5 to 7 mm below the bulbs of the follicles to reach minimal transection of hair follicles. To have optimal visibility, skin hooks on both sides of the ellipse were used for retraction. The ellipse was removed with scalpel or scissors. If needed, hemostasis was conducted by electrocautery. 2 based on blood supply in the area of scar. The patient’s hairline design was shown in front of a mirror.For graft site creation, various instruments were used including 19- and 20-gauge needles (South Korea), chisel and custom made blades to make sites as small as 0.6 and 0.7 mm in diameter. The scalpel blades used for 1-hair-grafts were 0.6 to 0.7 mm, while those used for grafts containing 2-hairs-, and 3-hairs-grafts were 0.9, and 1.0 mm. The strip was divided in slivers, and then, each sliver was divided into individual FUs of 1-, 2-, 3-, or 4-hair grafts depending on how they existed in the scalp. The donor region was repaired with sutures in a single plane with 4-0 nylon joining the edges carefully and precisely. Once the last suture was carried out to close the donor site, a temporary dressing was applied, and the patient turned over again. The sutures were removed after a week. To achieve acceptable density, we implanted between 20 and 40 units/cmThe graft placement involved two specialized nurses to move the hair efficiently into the graft sites using forceps with curved tips, straight tips, cotton-tipped applicator, and Choi implanter under good ergonomics and lighting. The grafts were continuously well hydrated in physiological saline solution throughout the entire process from extraction to implantation to improve the survival rate. In insertion of the grafts into the recipient incisions, the two registered and trained nurses worked together, 1 on either side of the patient, introducing the follicular grafts by straight or angled fine-tipped jeweler’s forceps and hair implanters into the incisions individually inserting about 10 units/min. Once the grafting was finalized, an overnight dressing was applied, and patients could return home. They were instructed to be cautious not to traumatize the newly placed grafts. The detailed timeline for expected hair growth were shared with the patient and were instructed for the medications including antibiotics, minoxidil, supplements such as zinc, 5% Rogaine  and platelet rich plasma (PRP) therapy two times per year. The patient was asked to be visited the following day. The frequency of further visits was dependent on the patient’s status and the presence of any probable complications that was weekly during the first month after the procedure followed by a 6-month interval.Fifty-six patients were enrolled in this study. Most of them were 31-40 yr old (48.3%), and the least were older than 50 yr old age groups . Male subjects consisted 71.4% of the patients. Trauma (55.4%) was the most causative agent responsible for scar alopecia and the least were radiotherapy (1.8%) and leishmaniasis (1.8%) registered in their medical history leaving scar mostly in in scalp (39.3%), and the least in moustache (10.7%) regions .The used surgical designs were FUE (87.5%), FUT (10.7%) and a combination of both surgical procedures (1.8%). When FUE was targeted for transplantation, scalp (83.7%) and beard (16.3%) were the harvesting donor regions. For scalp, the maximum number of grafts  was used and the least grafts in moustache , while 2-hair- and 3-hair-grafts mostly in scalp transplantations  as treatment options23,24. Successful outcomes have been reported with the use of tissue engineered dermal regeneration templates followed by follicular unit transplantation to reconstruct large scalp defects25,26. In our study, patients received minoxidil and if patients were willing, PRP was added as complementary treatment options.Currently in hair transplant clinics, the majority of patients are recommended for medical therapy to maximize the efficacy of any transplant, to stimulate hair growth and to enhance graft survival including minoxidil and finasteride27. Moreover, there are significant challenges such as limited donor supply, staff training, the length of time of the procedure, and developing new medications8,28. Our center was rich in technical skills and enrolled artistic abilities to create an appropriate site angling and hairline design that fitted with the patient’s overall features29. In our patients, there was the challenge of limited donor supply in scalp of some patients that was effectively compensated in male subjects by use of non-scalp body hair such as beard as potential sources of donor grafts. As hairs grow slightly laterally and then downward along the goatee, it is of great importance to make an angle as acute as possible to the skin16. The creation of density in this area was a little difficult in our center due to the topography of the upper lip’s cupid’s bow area. Therefore, a maximal density packing of 2-hair-grafts were used. When approaching eyebrow restoration, it is important to monitor the shape of masculine eyebrow versus feminine eyebrow appearance. As the masculine eyebrow shape is generally less arched, it comes to a lateral widening at the peak of the brow, whereas the feminine eyebrow shape is more rounded and arched and we tried to cover the patients’ expectations. No complication happened in our patients and they were all satisfied with the results. There could be some limitations in all studies using hair transplantation in scarring alopecia including complications encountered in hair transplantation surgery such as enlarged scars, folliculitis, keloid, necrosis neuralgias, donor hair effluvium, and arteriovenous fistulas in the donor areaTrauma and burn were demonstrated as the most prevalent causes of scarring alopecia in patients seeking for hair restoration and scar reduction. In head and neck scar alopecia, hair transplantation was selected based on type and depth of scar. FUE was targeted when huge grafts were not needed, including beard, moustache, and eyebrow, while FIT was used when extensive scars were present in scalp. One-hair-grafts were mostly applied for eyebrow, moustache and beard, 2-hair- and 3-hair- grafts for beard and scalp transplantation. These findings can be added to the literature when FUE, FIT, or their combination are targeted in hair restoration of scar alopecia in head and neck. The authors declare that they have no conflict of interest and they present their consent to publish the article.


Troponin levels can be elevated in various diseases other than acute myocardial infarction, including sepsis. In diseases without myocardial necrosis, the elevated troponin levels are relatively low and normalize quickly.A 61-year-old Japanese man with impaired consciousness was transported to our hospital. He was diagnosed as having pneumonia and septic shock. His condition was severe, but his clinical course was good. However, his troponin level remained extremely high during admission; on the second day, it was higher than the measurable range. We consulted a cardiologist and performed echocardiography and myocardial perfusion scintigraphy but found no new ischemic changes.In septic shock, troponin levels can be extremely high, which can persist even after recovery, as in very large myocardial infarctions. Troponin level is elevated in various diseases other than acute myocardial infarction (AMI), including sepsis. In these diseases, the elevated troponin level is relatively low; the higher the troponin, the greater the possibility of AMI . MoreoveStreptococcus pneumoniae) and septic shock, and acute upper gastrointestinal bleeding. The Acute Physiology and Chronic Health Evaluation II and Sequential Organ Failure Assessment scores were 54 and 12, respectively. In the emergency department, orotracheal intubation was performed, and antimicrobial administration, fluid resuscitation, vasopressor agent administration, and blood transfusion were started. After admission to the intensive care unit, continuous hemodiafiltration (CHDF) was also started for AKI. On the first day of admission, he had a pulseless electrical activity (PEA) twice, but the return of spontaneous circulation was achieved at both times with a 1 g adrenaline dose. CHDF was terminated on the second day, and vasopressors/inotropic agent administration was terminated on the third day. The patient was weaned off from the ventilator on the seventh day and transferred to the hospital for rehabilitation on the 23rd day. After the transfer, he had no problems and was discharged.A 61-year-old Japanese man with impaired consciousness was transported to our hospital. He had a history of type 2 diabetes mellitus, old myocardial infarction, angina pectoris, and chronic kidney disease (CKD). On initial evaluation, he had shock with cool peripheries, blood pressure of 79/68 mmHg, heart rate of 78 beats per minute, Glasgow Coma Scale score of 3/15, and axillary temperature of 31 °C. Additionally, transthoracic echocardiography (TTE) revealed diffuse left ventricular wall dyskinesia, with a visual ejection fraction (EF) of 20–30% . Electrocardiography revealed widespread ST depression with ST elevation in the aVR lead  showed that the median hs-cTnI levels (ng/L) in sepsis on the first through the third day were 109 [interquartile range (IQR) 39–394], 103 (IQR 38–449), and 79 (IQR 31–281), respectively [In this case, the high troponin level persisted for at least 20 days. In general, troponin levels decline relatively quickly in diseases other than AMI . HoweverThe present case had a good outcome even though the troponin level continued to be extremely high. A recent study showed that, in sepsis, the higher the hs-cTnI level, the higher the mortality rate, but the relationship disappeared when the level was > 100–500 ng/L . TherefoSCM alone cannot explain why the troponin level was extremely elevated in our case. The patient had a history of coronary artery disease and CKD. These are factors that cause elevation of troponin level in sepsis . MoreoveHerein, we report a case of extremely high hs-cTnI levels induced by septic shock. In spite of the high levels, the patient’s clinical outcome was good, and no evidence of new myocardial ischemic findings was found.


Despite tropical forests representing the most important terrestrial carbon stock globally, systematic examination of historical afforestation in these habitats in the Neotropics is lacking. Additionally, there has been no assessment of similar depopulation–afforestation dynamics in other parts of the global tropics that were incorporated into the Spanish Empire. Here, we compile and semi-quantitatively analyse pollen records from the regions claimed by the Spanish in the Atlantic and Pacific to provide pan-tropical insights into European colonial impacts on forest dynamics. Our results suggest that periods of afforestation over the past millennium varied across space and time and depended on social, economic and biogeographic contexts. We argue that this reveals the unequal and divergent origins of the Anthropocene as a socio-political and biophysical process, highlighting the need for higher-resolution, targeted analyses to fully elucidate pre-colonial and colonial era human–tropical landscape interactions.It has been suggested that Iberian arrival in the Americas in 1492 and subsequent dramatic depopulation led to forest regrowth that had global impacts on atmospheric CO The authors assess the impact of Spanish colonization on forest dynamics across the tropics, finding variable responses according to regional land use strategies, as well as other cultural, social and biophysical factors. Tropical forests, which cover only 14% of the Earth’s surface5 but contain 68% of the global living carbon stock6 and half of the Earth’s biodiversity7, are a central feature of this discussion8. Indeed, human-driven, habitat-scale reorganization of these systems  is thought to pose an “existential threat to civilization”10. The search for the beginnings of the Anthropocene has, in geological circles, centred on the identification of a single golden spike11. Attempts to track this critical transition initially focused on the onset of industrial fossil fuel burning in the eighteenth and nineteenth centuries4, but today concentrate on the Great Acceleration in the 1960s12. However, there have been growing calls in the social sciences to search for the origins of the Anthropocene as a long-term process that extends back into the pre-industrial era14, based on the premise that early agricultural processes substantially impacted atmospheric greenhouse gas concentrations, including CO2 levels3. This is particularly important within carbon-rich tropical forests where archaeological and palaeoecological research has revealed evidence for substantial human impacts on ecosystems, species distributions and soils over the past 45,000 years15. However, while there is increasing consensus that pre-industrial societies had large impacts on global ecosystems and biodiversity17, the exact scale and nature of anthropogenic alteration, particularly with respect to forest cover and CO2 concentrations, remains to be elucidated.The term Anthropocene—used to describe a new epoch in which human activity has become the dominant influence on Earth systems—has been vigorously debated in the natural and social sciencesce as part of the expansion of the Spanish and Portuguese empires (termed the Columbian Exchange18) resulted in the radical reorganization of life on Earth without geological precedent19. Not only did Iberian colonizers bring new crops, animals and ways of using the land to the tropics18, they also introduced lethal diseases from Eurasia. The ensuing pandemics, alongside starvation and murder, wiped out up to 90% of Indigenous populations in the Americas, with the impact of their lack of immunity compounded by colonial policies focused on urban relocation and enslavement21. Earth system scientists have argued that this Great Dying, and the abandonment of traditional land use now known to have been extensive across the Neotropics, was so widespread that it led to dramatic forest regrowth23. The latest estimates suggest that subsequent afforestation captured 7.4 PgC  from the atmosphere, resulting in a CO2 level drop recognizable in ice cores by 1610 ce and driving global cooling seen in the form of the Little Ice Age (LIA)21. Although the global impacts of this regional signal have been promoted as a potential golden spike for the Anthropocene and the scale of the Great Dying is historically well documented (noting that specific estimates remain debated), direct evaluation of consequential vegetation change and overhaul of land management in carbon-rich tropical forests following Iberian colonization has been limited. Research of this nature in the Americas has often been locally constrained , while larger-scale analyses have focused on the synthesis of charcoal records to reveal population and land use change, including a sustained period of reduced biomass burning after ~500 calibrated years before the present 26. Assessment of ecosystem responses to these demographic and land use drivers has, to date, been qualitative, non-systematic and focused on a small number of datasets21. Homogenous, broad-scale ecological transitions in response to depopulation in the Neotropics thus remain more assumed than proven.It has been proposed that contact between the so-called Old World and New World after 1492 18, discussed impacts at a global scale, recent framings of this phenomenon on tropical ecosystems and their Earth systems feedbacks have been almost entirely limited to the Atlantic sphere21. This is despite the fact that, following their arrival in the Philippine archipelago in the sixteenth century, the Spanish Empire (including Portuguese-claimed regions incorporated into the Spanish Empire between 1580 and 1640 ce during the short-lived Iberian Union) united Madrid, Mexico City and Manila into the first truly pan-tropical biological, cultural and economic system27 —particularly those that were geographically isolated from Eurasia—witnessed large-scale  disease spread29 and the introduction of additional novel domesticates30 between the 1500s and 1700s   curvature and in the plant functional grouping ) for two of the 11 sites, which are located within the Los Llanos tropical savanna (Laguna Mozambique37) and Andean valley dry forests (Quilichao basin38) . This spatial response bias likely reflects the cooler, wetter conditions associated with the LIA in the Andes, coupled with the sensitivity of seasonally dry and Neotropical montane forests to changing climate drivers41. There is no clear spatial or cultural relationship among the remaining eight records that reflect pre-Iberian afforestation. However, five of these sites occur within biomes other than moist tropical forest . This implies heightened climatic sensitivity of non-rainforest biomes that lie closer to precipitation thresholds and/or that changes in the availability of resources under a changing climate regimen within these habitat types encouraged social restructuring. Two of the 28 records indicate forest opening between 1500 and 1600 ce: Lake Caranã42  and Cenote San Jose Chulchaca43 .Eleven of the 28 records show a degree of afforestation between 1500 and 1600 ce . There ce Fig. .36 45) and 75% of the records are located within seasonally dry tropical forest  is also reflected in a charcoal record from Pohnpei46, although in this case there is no evidence of corresponding afforestation, possibly due to the simultaneous introduction of pigs to the island by the Spanish , while the Lake Paoay48 (Philippines) charcoal data do not include the Spanish colonial period —which also appear to have relatively low post-colonization mortality rates  and charcoal (6/10) records show afforestation and decreased fire in the landscape in the centuries before Iberian imperial influence. There is no consistent geographic or ecoregional pattern associated with this signal. This suggests that changes in forest cover after colonization were, in many cases, muted relative to those caused by land use or climatic factors before Iberian contact.The lack of sampling resolution in the Asia-Pacific records, as well as the relatively late influence of Europeans on some of the islands , Yap and Pohnpei (1700s); Supplementary Text 21. Furthermore, it provides the first evidence that this process was not exclusive to the Americas, but also occurred in the Spanish East Indies  appears to have resulted in a geographically isolated settlement and/or a protracted Iberian settlement process, thereby drawing out the spread of Eurasian pathogens and land use  following Iberian contact. This, in part, appears to support claims made in previous work that the documented decrease in Indigenous populations in the Americas following the introduction of foreign diseases and colonial policies led to a collapse of existing farming and food production systems and concomitant forest regrowth60. Similarly, islands that were seemingly pushed towards their natural resource limits by pre-Iberian populations  are, from a biogeographic perspective, more sensitive to disturbance61 and appear to show a higher prevalence of forest regrowth after European contact. Interestingly, the more open habitat types that appear to show the greatest forest dynamism in response to Iberian conquest have, in general, lower carbon sequestration capacity than the apparently less sensitive, dense, perpetually humid rainforests of South America or Southeast Asia6—a factor that is overlooked in the calculation of the impact of early colonial era afforestation on the global carbon budget21. It is also worth noting that our dataset lacks coverage within key pre-Iberian and Iberian era urban hubs—regions that would be expected to show higher levels of ecological restructuring following colonization. Key under-researched sites include the Valley of Mexico (controlled by the populous Empire of the Triple Alliance at the time of Spanish contact) and important Spanish settlements in the Philippines . A lack of research within important Iberian hubs relates, at least in part, to the fact that most pollen-based studies focus on reconstructing past ecological–climate or human–environment relationships over much longer time scales, biasing site selection away from landscapes that have been heavily modified over the past ~1,000 years40. Targeted site selection to European settlement and trade centres, as well as improved chronological and sampling control within recent centuries, is thus an important element of future palaeoecological work. Indeed, these limitations have been raised in the context of recent work attempting to use palaeoecology to gauge pre-European landscape burning patterns in Northern America62.Ecological and biogeographic factors may also have mediated forest resilience to human disturbance in both the pre-colonial and early colonial era. For instance, seasonal ecosystems within both the Atlantic and the Pacific appear more prone to Iberian-era afforestation, potentially reflecting their structural reliance on Indigenous land use practices, particularly swiddeningce). In several cases, this process actually appears to have exceeded early Iberian era forest regrowth in terms of scale. Notably, it suggests that coupled atmospheric–human drivers 64, as well as the heterogeneous expression of the Medieval Warm Period (MWP) and LIA across the study region , as well as in the resource-limited Pacific islands, than in perpetually humid forests. However, it is also worth pointing out that climate-driven landscape changes were, in all likelihood, accompanied and potentially reinforced by human behavioural adaptations. For instance, there is evidence that Indigenous groups, including the Classic and post-Classic Maya and Inka, engaged in adaptive agroforestry and developed new agricultural practices to cope with climatic extremes66. Similarly, the ENSO-driven 1300 ce climate event in the Pacific, which resulted in a substantial depletion of available food resources  highlight that industrial era deforestation far exceeds in magnitude any other shifts in forest cover over the past 2,000 years. The low temporal (subsampling) resolution of core tops and the spatial sampling bias towards sites surrounded by more intact ecosystems means that this change is likely to be under-represented in the assessed pollen records.Our data also demonstrate patterns of deforestation after Iberian arrival, both as a more immediate response to settlement and as a later response to the broader consequences of European colonialism, including the rise of capitalist European hegemonyent Fig. . A poten21, as well as the Asia-Pacific73, the timing and extent of observed afforestation in the early Iberian era appears contingent on spatially variable cultural and climatic factors coupled with ecoregion-specific resilience. Tropical forests reflect long-term land use legacies on an interhemispheric basis15. The murder, relocation and infection of Indigenous populations in many regions, as well as the floral and faunal exchanges that took place following Iberian colonialism, are essential considerations for re-evaluating the Anthropocene as a temporally variable and biogeographically/culturally contingent unequal process13. However, the variations in forest dynamics we observed before and after the initial period of Iberian contact and the establishment of colonies highlight the need to develop more detailed records of vegetation and land management change in different parts of the tropics, combining archaeology, palaeoecology and Indigenous traditional knowledge. This will permit a comprehensive exploration of the ways in which Indigenous resistance, invasive species, economic imbalances and the extension of colonial power and profit-driven land use left their varied marks on contemporary landscapes around the tropical world. Addressing these fine-scale, interdisciplinary questions will require well-dated, high-resolution palaeoenvironmental reconstructions spanning the past 2,000 years and covering the range of pre-colonial and colonial land use strategies that were present across the tropics. Only when such records become available can more realistic estimates of land use change and corresponding carbon fluxes be produced and fed into Earth systems models, with current projections21 likely to be simplifications. It is also clear that more refined understandings and records will enable conservation practitioners to grapple with the diverse socio-political, cultural and economic factors that have shaped, and continue to shape, the composition, diversity and resilience of tropical landscapes into the twenty-first century.Overall, our analysis indicates that while forest regrowth did often occur following the decimation of large Indigenous populations after Iberian contact in the tropical Americasbp26. In some instances, this change has been linked to a decrease in anthropogenic fire use following Iberian arrival and has been used to support the hypothesis that reduced land cultivation following a decrease in the population led to region-wide afforestation. However, site-specific discussion of the role of changing fire regimens relative to vegetation response are discussed in Supplementary Text 26, which shows a decrease in charcoal influx (biomass burning) between 1500 and 1650 ce to a minimum at 1650–1700 ce, is included in Fig. Due to the inaccessibility of raw palynological data from sites in the Spanish East Indies relative to the Spanish Americas , which were relevant for reconstructing tropical floristic change in the former Spanish Americas before, during and after Spanish colonization, using the following criteria:The record was located within geopolitical units that were part of the former Spanish Empire (including Brazil).The record was directly dated.ce, permitting reasonable assessment of the scale of Iberian-induced change relative to the past 2,000 years. These datasets  to a tropical or subtropical biome.The record derived from a terrestrial site that currently occurs within a tropical or subtropical biomece, thereby permitting assessment of floristic response to any Iberian-induced land use change.The record included one sample that was estimated to come from the time frame 1500–1600 bp) was <200 years per sample.The temporal resolution of the upper 2,000 years of the record . An overview of how these events impacted the various geographic regions assessed in this study is outlined in Supplementary Text The cut-off in criterion (6) was set in an attempt to capture forest turnover while maintaining a reasonable distribution of records across the study area. The Cobweb Swamp (Sawgrass Core)The application of the above criteria resulted in a final selection of 28 pollen records (from the same number of sites) for analysis. The setting, location and publication(s) associated with the selected records are detailed in Supplementary Data 77. While this is problematic for many Neotropical records78, our selection of the pollen records based on sampling density and chronological control over the time period of interest attempts to selectively remove data that do not adequately capture ecosystem dynamics before, during and after the Iberian colonial period. We used the most up-to-date chronological models developed for the records in Neotoma and include a discussion of the interpreted ecological change, in the context of each chronological model produced for the records, as part of Supplementary Text The chronology and sampling resolution of the pollen records can influence whether rapid response dynamics are captured and appropriately constrained79. Once regrouped, we converted raw values to relative abundances and plotted each dataset stratigraphically against the age–depth models produced for each record  is the variability in the taxonomic resolution and the range of methods used by the original authors to classify vegetation change through time. To manage this, we adopted a two-pronged approach to assessing ecological change. First, we used the raw pollen counts from each record and consistently reclassified all individual pollen taxa into nine plant functional groups that can provide information about major state shifts in site vegetation through time. These functional groupings are listed in Extended Data Fig. 84 were searched for pollen and charcoal data within the time frame 0–2000 ce from countries within the former Spanish East Indies. This search returned four charcoal records . The Lake Paoay pollen data were prepared by calculating and plotting the ratio of grass to arboreal pollen as a proxy for forest openness. This proxy was chosen to maintain consistency with other available pollen curves from the region.Neotoma and the Global Paleofire Database85. While all efforts were made to ensure precise data extraction, minor sample or variable offsets may have been introduced as errors into the dataset depending on the quality of the initial graph production. Because some of these plots were made against depth (versus age), and the interpretation of age was based on outdated chronologies, an updated age–depth model for several of the cores was constructed using the program Bacon86 (R version 3.6.2)81 , as well as any complimentary charcoal data, were extracted using WebPlotDigitizerThe extracted data totalled ten pollen records, eight charcoal records and one phytolith record from 13 sites. Site details are outlined in the Supplementary Data 88. Data were first standardized by transformation  and then standardized into z scores. Each GAM was fitted using thin-plate regression splines as the basis function89: the rank of the basis function was set to one-tenth the sample size or 5, whichever value was larger (ranks ranged from 5–26). GAM plots included 95% uncertainty intervals around the GAM fit line. Implementation of the GAM fits, calculation of uncertainty intervals and creation of record-specific GAM plots was undertaken in R version 3.6.2 (ref. 81) using the mgcv package (version 1.8-31)90. For data importation and manipulation, we used the packages data.table (version 1.12.8)91 and readxl (version 1.3.1)92. Resultant data are plotted stratigraphically in Fig. https://osf.io/gu483/, which also includes code to apply the same methods used in this paper to other datasets.The non-arboreal to arboreal ratios calculated for each Neotropical record, and the charcoal data and various pollen proxies for forest openness in the Spanish East Indies, were summarized using GAMs. This permitted an assessment of nonlinear trends in palaeoecological data, particularly those that are irregularly spaced in time, as is the case both within and between some of the Neotropical pollen datasetsce) afforestation or limited forest change over the past 1,000 years. We set 1,000 years as the time frame for classifying change as it is sufficiently long to provide a context for the pre-Iberian forest conditions while reducing the need to consider the influence of protracted mid- to late Holocene climate change on forest cover40. However, attention was given to the timing and asynchronous influence of shorter-term climate events on forest cover over the past 1,000 years , the regional influence of which is discussed in Supplementary Texts Post-Iberian afforestation : the forest cover proxy data and GAM curvature show an increase in forest pollen in the 100-year time frame following a known population decrease associated with Iberian contact. For the Neotropics only, the clustered PFG data show that this shift is associated with a clear change in forest type and forest cover.Minor (or unclear) post-Iberian afforestation: either (1) the forest cover proxy data and GAM curvature show an increase in forest pollen in the 100-year time frame following a known population decrease associated with Iberian contact but the PFG data indicate forest stability over the same time period or (2) the forest cover proxy and PFG data indicate an increase in forest pollen in the 100-year time frame following a known population decrease associated with Iberian contact but this is not captured in the curvature of the GAM.Post-Iberian deforestation : as for post-Iberian afforestation, but the data indicate forest opening rather than closing.ce for the Neotropical sites or between 1000 ce and the timing of Iberian contact for the Asia-Pacific sites.Pre-Iberian afforestation: there is an afforestation or minor afforestation signal (as above) in the period between 1000 and 1400 A limited forest response was determined if the records did not meet any of the above-listed criteria.The palaeoecological proxies for forest cover, their associated GAMs, the known timings of Iberian contact and disease-induced population decrease  after Iberian contact that were not captured in the GAM curvature were classified as minor/uncertain.Site-by-site analysis of the palaeoecological and chronological trends and confidence for each record, together with other external supporting data, are presented in Supplementary Text The results of pre- and post-Iberian land use and forest change analysis were mapped for each region in ArcGIS Pro 2.5 Fig.  and inteFurther information on research design is available in the Supplementary InformationSupplementary Text 1–3, Figs. 1–34, Tables 1–3 and References.Reporting SummarySupplementary DataDetails of the individual records used in this study. Supplementary Data 1: list of the Neotropical (Spanish American) records used in this study, including Neotoma codes, source, location and site details. Greyed-out records include the studies that did not meet the criteria for inclusion in our analyses . Supplementary Data 2: list of the Asia-Pacific (Spanish East Indies) records used in this study, including location, site and publication details.


Bipolar and schizoaffective disorders are both psychiatric illnesses that share common traits, but also significant differences. Due to the close overlap in symptoms, obtaining the correct diagnosis can be difficult. The management of these patients often poses a challenge to clinicians.Five years ago, our patient was misdiagnosed with bipolar disorder with psychotic features. It was later discovered that she was suffering from schizoaffective disorder, bipolar type. The schizoaffective disorder involves symptoms of both schizophrenia and mood disorder. Bipolar and schizoaffective disorders share common characteristics and because of the overlap in symptoms, getting an accurate diagnosis can be very arduous for clinicians. A comprehensive plan of previous treatment results, psychiatric and family history can help in the differentiation between these disorders .Bipolar disorder is a debilitating condition and its global prevalence ranges from 1.4% to 6.4%, and individuals may experience episodes of mania, depression, and sometimes, psychotic symptoms . The schA cross-sectional study was held with a majority of patients suffering from psychiatric problems such as bipolar, schizophrenia, schizoaffective, and other depressive disorders. This study concluded that 39.16% of patients with psychiatric disorders were misdiagnosed, with schizoaffective disorder having the greatest rate of incidence of misdiagnosis at 75%, and bipolar disorder at 17.78% .Our patient was misdiagnosed with bipolar disorder with psychotic features. On admission, with proper history taking and examinations, she was diagnosed with schizoaffective disorder, bipolar type. There are major parallels between these two disorders, but we must learn to identify key differences to provide appropriate care and treatment. Attempts have been made to better understand the incidence of misdiagnosis.A 50-year-old hypertensive, hyperglycemic, African American female presented with a history of bizarre delusions, hallucinations, disorganized behavior, and disorganized speech for the past one and a half months. The patient claimed she found three homeless in one of her beach homes, who attacked her, placing her in the hospital. She was unable to explain why she was in the psychiatric unit. On further investigation, the patient stated that she was 10 years old, a celebrity and that her father was the president of the United States. She denied any depression, homicidal, or suicidal ideations. Her past medical history was significant for diabetes type 2, hypertension, epilepsy, HIV, and acute myeloid leukemia, which are now in remission. She was previously diagnosed with bipolar type 1 disorder with psychotic features.On physical examination, her vitals were stable and there was a scar on her right knee. When asked, the patient said the scar on her knee is a fresh wound that she got when she was attacked, before she was admitted to our hospital. When examined, the scar had properly healed, but the patient still thought she was actively bleeding. On mental status examination, her appearance was unkempt and she was poorly groomed. Her behavior was childish and very cooperative. She could name, read, repeat and comprehend, but was unable to write a complete sentence. The patient was oriented to time and place, but not to person or situation. She was euphoric and her affect was appropriate to the content. Her thought content included grandiose delusions and an overabundance of thought. Her thought process consisted of loosening of associations, flight of ideas, and tangentiality.At the time of initial presentation, the patient was manic, easily distracted, and demonstrated aggression for not believing her. She also disrobed in front of staff and patients (indiscretion), had grandiose delusions that she was a 10-year-old child celebrity, believed she was the daughter of the American president, exhibited flight of ideas, psychomotor agitation, and pressured speech. These symptoms were also observed by the nursing home she resided in prior to her admission to the psychiatry unit. She was subsequently diagnosed with schizoaffective disorder, bipolar type according to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-V) criteria. The patient had delusions, hallucinations, disorganized speech, and she also exhibited grossly disorganized behavior for more than a month. These hallucinations (burglars attacking her in her beach home and seeing blood on her knee), delusions, disorganized speech, and grossly disorganized behavior were present for two weeks in the absence of her mood symptoms. Her mood episodes were reported to be present for the majority of her illness, which is why her disorder was confused with bipolar disorder with psychotic features instead of schizoaffective disorder, bipolar type. Our patient’s psychopharmacological treatment included Sertraline 50 mg once daily, Quetiapine 100 mg PO QHS, and Valproic acid 500 mg PO BID.The patient was treated with the combination therapy of an antipsychotic with a mood stabilizer and an antidepressant. This combination therapy is used to treat 18% of schizoaffective patients. Paliperidone is the antipsychotic of choice approved by the FDA for schizoaffective disorder, but due to financial limitations, paliperidone could not be used in the treatment of our patient . QuetiapThe diagnostic criteria of schizoaffective disorder overlap greatly with many other severe psychiatric disorders seen in practice, which has made schizoaffective disorder one of the most frequently misdiagnosed psychiatric disorders . CharactThe continuous synchronization of psychosis and mood episodes makes it hard to distinguish mood disorders with psychotic features from schizoaffective disorder and bipolar type ,7. ThereIn our case, the patient was misdiagnosed with bipolar disorder with psychotic features. After a thorough history and examination, she was diagnosed clinically with schizoaffective disorder, bipolar type. Hallucinations and delusions are prominent symptoms of schizoaffective disorder. Another symptom of schizoaffective disorder is disorganized thinking and disorganized behavior. Furthermore, for schizoaffective disorder, bipolar type, there may be periods of both depression and mania. All these symptoms were present in our patient.Attempts have been made to better understand the incidence of misdiagnosis. The more contemporary survey conducted by National Depressive and Manic-Depressive Association (DMDA) inferred that 69% of patients with bipolar disorder were initially misdiagnosed, and more than 33% of patients remained misdiagnosed for another decade before attaining an accurate diagnosis . FriedriAn accurate history and physical examination are paramount to correct diagnosis, during which schizophrenia, a major depressive disorder with psychotic features, and bipolar disorder must be ruled out during the workup . An obseSchizoaffective disorder can be arduous to manage due to the manifestation of both psychotic and mood symptoms. A study by Cascade et al. had reported that 87% of patients from the SDI/Verispan’s Prescription Drug & Diagnosis Audit (PDDA) database with schizoaffective disorder, had received medications from two or more pharmaceutical classes between January 2008 and December 2008 . Also, 9It was reported in another article by Schnitzer et al. that combined treatment with an antipsychotic and antidepressant is an acceptable form of initial treatment for patients who have been diagnosed with schizoaffective disorder, depressive type . For patSeveral factors including an early administration and extended duration of antipsychotic medications such as paliperidone and aripiprazole have also been found to enhance the overall functioning of patients with schizoaffective disorder and schizophrenia . ClozapiSchizoaffective disorder, bipolar type, is frequently misdiagnosed in clinical practice. Psychiatrists believe that it can be avoided by getting a detailed history and proper examination in all patients with psychotic symptoms to differentiate between schizophrenia, bipolar disorder, and schizoaffective disorder. Our patient remained misdiagnosed for five years with bipolar disorder with psychotic features before reaching the correct diagnosis, due to symptoms that overlapped with other severe psychiatric disorders. In our case report, we emphasize the importance of proper examination and history taking in patients presenting with readmission and overlapping symptoms.


Spermatogenesis, which is a continuous process from undifferentiated spermatogonia to spermatozoa in the seminiferous tubules, declines with age. To investigate changes in spermatogenesis with aging, we reconstructed the seminiferous tubules of 12 mice aged 12 to 30 months from serial sections and examined age-related and region-specific alterations in the seminiferous epithelium and spermatogenic waves in three dimensions. The basic structure of the seminiferous tubules, including the numbers of tubules, terminating points, branching points, and total tubule length, did not change with age. Age-related alterations in spermatogenesis, primarily assessed by the formation of vacuoles in Sertoli cells, were detected in the seminiferous tubules at 12 months. The proportion of altered tubule segments with impaired spermatogenesis further increased by 24 months, but remained unchanged thereafter. Altered tubule segments were preferentially distributed in tubule areas close to the rete testis and those in the center of the testis. Spermatogenic waves became shorter in length with age. These results provide a basis for examining the decline of spermatogenesis not only with aging, but also in male infertility. Seminiferous tubules develop from the fetal testis cords and consist of a seminiferous epithelium, which is divided into 12 stages in mice according to the cell association pattern9. Adjacent stages are aligned along a seminiferous tubule, and the time required for a particular stage to reappear in the same area is called the cycle, while the space occupied by a series of adjacent stages, including all possible types, is called the wave10. Spermatogenesis is a lifelong process but its efficiency, such as daily sperm production, declines with age.Spermatogenesis is a continuous process from undifferentiated spermatogonia to spermatozoa in the seminiferous tubules12. The impact of aging on semen parameters, such as sperm motility, sperm morphology, sperm DNA integrity, telomere length, and epigenetic factors, has been extensively examined14. However, the impact of aging on the testis structure remains unclear. The major morphological and functional alterations that occur in the testis with age include the formation of vacuoles in Sertoli cells, breakdown of the blood–testis barrier, thickening of the basement membrane, decrease in the number of Sertoli cells and spermatogonia, arrest of spermatocytes, formation of multinucleated spermatocytes, premature release of spermatids, and accumulation of lipofuscin granules in Leydig cells23. To the best of our knowledge, a three-dimensional (3D) analysis has not yet been performed on the structure of aged testis and it currently remains unclear whether the alterations described above occur preferentially at specific sites in the testis.The decline in spermatogenesis with age is gradual and the complete cessation of reproductive capacity never occurs28. We detected region-specific morphological and functional differences within the testis. For example, we revealed that the sites at which spermatogenesis initially occurred in the postnatal mouse testis were preferentially distributed in areas of tubules close to the rete testis25, and that the extent of markedly impaired spermatogenesis was significantly greater in areas of tubules near the branching points in mice administered busulfan26. Furthermore, we performed an analysis of the waves, which is difficult using only 2D histological sections, and identified 76 waves with an average length of 16.9 mm in one adult mouse testis25. The biological meaning of the presence of waves may reside in the continuous and efficient production of spermatozoa without time intervals.Our group recently reported the high-resolution 3D structure of all seminiferous tubules in animal testes using serial sectionsIn the present study, to examine changes in spermatogenesis with aging, we reconstructed all of the seminiferous tubules of C57BL/6 mice aged 12 to 30 months from serial sections and performed a 3D analysis of age-related and region-specific alterations in the seminiferous epithelium and waves.Twelve mice were divided into 4 groups of 3 mice each according to age: 12 , 18 , 24 , and 30  months postpartum. All serial sections were stained with periodic acid-Schiff-hematoxylin (PAS-H) and all seminiferous tubules were reconstructed in 12 testes, one from each mouse. The results of 4 representative testes, one from each group, are shown in Fig. The seminiferous epithelium consists of germ and Sertoli cells, with the latter exhibiting almost no proliferative or regenerative capacity in the adult testis. Therefore, to examine age-related alterations in the seminiferous epithelium, it is reasonable to assess the presence of vacuoles formed in Sertoli cells. All seminiferous tubule areas observed in each testis section were classified into two types according to the cell association pattern and the presence or absence of vacuoles formed in Sertoli cells: normal (green), normal spermatogenesis and the absence of vacuoles; altered (red), the presence of vacuoles Fig. e–j and/oWe examined whether the degree of alterations in the seminiferous epithelium differed among different 3D positions in the whole testis and in individual seminiferous tubules. A quantitative analysis of the 3D distribution of altered segments was performed in 12 testes, one from each mouse with the rete testis situated at the upper left corner, as in Fig. To analyze age-related alterations in spermatogenic waves, the stages of all seminiferous tubules in each section were assessed in #2 and #10 testes representing 12 and 30 months, respectively. All 12 stages were identified in PAS-H-stained sections based on the cell association pattern and the shape of acrosomes stained with PAS. The stages of the seminiferous epithelium are generally aligned in a descending manner from the rete testis side. However, this basic pattern of the arrangement of stages is often interrupted by local irregularities in the order of stage numbers. Furthermore, individual stages are too short in length for a 3D analysis. Therefore, to avoid complexity, we divided the seminiferous epithelium into 3 groups, i.e., stages I–VI, VII–VIII, and IX–XII. In addition, there were degenerated segments, the stages of which were unclear because of severely impaired spermatogenesis, as shown in Fig. 29, the present results are consistent with previous finding showing that the decline in spermatogenesis with age is gradual and the complete cessation of reproductive capacity never occurs12. Additionally, aging did not alter the basic structure of the seminiferous tubules, including the numbers of tubules, terminating points, branching points, and total tubule length.The present study revealed that the extent of age-related alterations in the seminiferous epithelium represented by vacuoles formed in Sertoli cells, which were already found at 12 months, increased significantly by 24 months, but remained unchanged thereafter. Even at 30 months, more than 60% of areas in the seminiferous epithelium retained a normal morphology. Since the average lifespan of C57BL/6 mice is approximately 28 months30. This structure is different from other seminiferous tubules: TR consists of modified Sertoli cells that proliferate, shows distinct functions, and is potentially vulnerable to inflammation. Moreover, immune cells are abundant in TR31. Therefore, chronic inflammation may promote region-specific alterations in the areas of seminiferous epithelium close to the rete testis with age. On the other hand, excess heat may promote region-specific alterations in the seminiferous epithelium in the center of the testis with age. Excess heat on the testis causes germ cell apoptosis and Sertoli cell dysfunction33. Temperature may increase more frequently in the central regions of the testis than in the surface regions beneath the tunica albuginea, because the latter are more efficiently cooled with open air than the former. The difference in the proportion of the altered epithelium between the surface and central regions of the testis became smaller at 30 months than at 12 months, suggesting that alterations in the seminiferous epithelium due to excess heat proceeds toward the surface regions with age. The surface regions of the testis mainly consist of cranial hairpin turns of seminiferous tubules, which had higher proportions of the normal epithelium than the caudal hairpin turns occupying the central regions of the testis. Therefore, the zig-zag convoluted structure of seminiferous tubules may be a mechanism by which the tubule portions close to the tunica albuginea are distributed evenly along the lengths of all seminiferous tubules and may maintain the integrity of individual tubules.The present study clearly demonstrated that the altered epithelium was preferentially distributed in areas close to the rete testis and the center of the testis. The area involving the junction between the rete testis and a seminiferous tubule forms a short special structure called the transition region (TR)34. The preservation of the wave is important for the maintenance of male fertility because the preservation of asynchronous germ cell differentiation constantly supplies numerous spermatozoa throughout life. The present study revealed that the segments become shorter and, as a result, the waves become shorter in length and larger in number with age. This phenomenon may be interpreted as the weakening of intercellular communications, including one with intercellular bridges, preventing the formation of large segments with age. This appears to be supported by the observation of sites at which non-adjacent stages were aligned such that the definition of waves is impossible at 30 months.The wave is defined as a series of adjacent tubule segments that contains all 12 stages in mice. A tubule portion occupied by an epithelium belonging to one of the 12 stages designated with roman numerals forms a segment. When a seminiferous tubule is traced from the rete testis, each segment is followed by a segment with the next smallest stage number. This observation suggests that the rete testis or other segments affect the stage of the adjacent segments26. By applying the present method, it may be possible to determine the specific patterns of the distribution of altered tubule portions in various types of spermatogenic disorders, including human infertility. The current limitations in applying the present study to the clinical purposes include the difficulty in obtaining the specimens from infertile human males, and the time and labor required for the 3D reconstruction that prevents the analysis of human testes with extremely large sizes.The region-specific alterations in spermatogenesis in aged mice, as revealed in the present study, are different from those in mice with busulfan-induced spermatogenic disorderMale C57BL/6 mice were purchased from Nippon SLC, Inc. , reared under standard 12-h light/12-h dark laboratory conditions with free access to standard food and water, and used at different ages: 12, 18, 24, and 30 months postpartum. All animal experiments complied with the ARRIVE guidelines. All procedures were approved by the Animal Care Committee of Kanazawa University  and were performed in accordance with relevant guidelines and regulations.35. Sections were digitized using a whole-slide scanner  with a 20-fold objective lens, and the resulting digital images of the sections were visualized with viewer software .Animals were sacrificed by cervical dislocation. The testis and epididymis were dissected out en bloc, fixed in Bouin’s solution overnight, dehydrated in a graded ethanol series, and embedded in paraffin. Five-µm-thick serial sections with intervals of 50 µm were cut using a microtome and mounted on glass slides. Sections were treated with PAS-H to stain the basement membrane of seminiferous tubules, as previously described28 with slight modifications. Briefly, extraction of the basement membrane stained in red–purple with PAS-H, which represents the outline of each seminiferous tubule, was performed in digital images using ImageJ software  and/or Adobe Photoshop 2020 software . After extraction, images were converted into gray scale in the JPEG format with Adobe Photoshop 2020 software at a resolution of 2724 nm pixel−1. Using Amira 6.3.0 software , serial images were automatically aligned followed by manual adjustments, and the inside of the outlines of a selected tubule was filled with a particular color using threshold processing and traced from section to section. This procedure, called segmentation, was repeatedly applied to all seminiferous tubules with different colors, and they were then 3D reconstructed. The core lines of individual seminiferous tubules were drawn using the same software. The whole testis was also 3D reconstructed by filling the inside of their outlines by threshold processing. The position of the rete testis was defined as the mean coordinate of the connections of all reconstructed seminiferous tubules with the rete testis and shown with a black sphere.The 3D reconstruction was performed as previously described12. The seminiferous epithelia of all seminiferous tubules in each section were assessed by histological observations using NDP.view2 and classified into two types according to the cell association pattern and the presence or absence of vacuoles: a normal epithelium with normal spermatogenesis and the absence of vacuoles; an altered  epithelium with the presence of vacuoles and/or impaired spermatogenesis. According to this typing, the core lines of all reconstructed seminiferous tubules were divided into segments of two colors. To analyze age-related alterations in spermatogenic waves, seminiferous epithelia were classified into 12 stages according to the germ cell association pattern by histological observations using NDP.view2 and the core lines of all reconstructed seminiferous tubules in 2 testes were divided into segments of different colors representing the 3 groups, i.e., stages I–VI, VII–VIII, and IX–XII. Furthermore, a wave was identified as a tubule segment containing successive stages of all 12 types. Waves were successively identified from the terminal points of tubules with the rete testis and marked on the core lines.The appearance of vacuoles in the seminiferous epithelium is the first visible age-related change at the light microscopic levelData were presented as the mean ± standard deviation (SD). Comparisons between two values were performed with the Mann–Whitney U test, and comparisons among multiple values were performed with the Kruskal–Wallis test followed by post hoc Dunn's test, with differences with a P-value of less than 0.05 being acknowledged as significant. Statistical analysis was undertaken using GraphPad Prism version 7.0e .


Background: Tick-borne encephalitis (TBE) is inflammation of the central nervous system (CNS) caused by a viral infection which may be associated with increased synthesis of immunoglobulins. It can lead to inter alia, breakdown of the blood-brain barrier (BBB), or even death and, unfortunately, treatment is only symptomatic. Therefore, the aim of the present study was assessment of the concentrations of free light chains (FLC) kappa (κ) and lambda  and serum of patients with TBE. Methods: A total of 58 cerebrospinal fluid and serum sample pairs were analyzed. Samples were collected from patients with TBE before and after treatment. FLC were measured using the turbidimetric method. The values of κIgG-index, λIgG-index, κFLC-index and λFLC-index were calculated using relevant formulas. Results: Pre-treatment serum λFLC concentrations were higher in comparison to post-treatment levels. Moreover, it was observed that CSF λFLC, TBEV IgM, TBEV IgG, and serum TBEV IgG, as well as the values of λFLC-index, κFLC-index, and λIgG-index were elevated after treatment. In the total study group, the concentrations of CSF κFLC and λFLC, and values of four indexes: κFLC-index, λFLC-index, κIgG-index, and λIgG-index correlated with each other and with CSF TBEV IgM and IgG antibodies. The CSF level of TBEV IgG was also associated with serum IgG TBEV and CSF IgM TBEV antibodies. Additionally, serum κFLC correlated with serum and CSF λFLC. Conclusion: This is the first study that demonstrates statistically significant differences in serum and CSF λFLC, as well as in the calculated values of three algorithms: λIgG-index, κFLC-index, and λIgG-index prior to and following treatment of TBE. Our findings may indicate that these differences reflect the intrathecal synthesis of immunoglobulins and increased permeability of BBB in patients with TBE. Moreover, it could provide the basis for developing new therapeutic strategies. The simplest definition of encephalitis is “brain inflammation”. The most common causes of encephalitis are viral infections . Tick-boThe diagnosis of TBE is based on the identification and determination of TBEV-specific antibodies. Serological examination of the serum and cerebrospinal fluid (CSF), obtained by lumbar puncture, is performed. Inflammatory changes in CSF last for a few weeks, and less frequently, several months. The concentration of TBEV-specific IgM and IgG antibodies in the serum and CSF is measured, and the results are compared with the diagnostic threshold value. The production and presence of specific IgM antibodies, usually lasting up to six months, is characteristic of the acute phase of the disease. Specific IgG antibodies persist for years and prove the obtained resistance to TBEV ,7. FurthCurrently, there is no causal treatment for TBE. Even severe forms of the disease are treated only symptomatically with anti-inflammatory drugs that reduce brain swelling, antipyretics, and painkillers . There aPatients admitted to the Department of Infectious Diseases and Neuroinfections at the Medical University of Bialystok constituted the study group. The participants underwent a lumbar puncture for diagnostic purposes. A total of 58 cerebrospinal fluid and serum sample pairs were collected from 29 patients with TBE . A total of 29 paired samples were obtained upon admission  and upon discharge—following 15.9 ± 6.2 days of hospitalization . On the basis of inflammatory parameters in the CSF with no focal neurological symptoms, thirteen patients were diagnosed with meningitis. On the basis of inflammatory parameters in the CSF, altered consciousness and, presence of focal neurological symptoms, sixteen patients were diagnosed with meningoencephalitis. Patient characteristics are presented in The diagnosis was based on clinical data and presence of inflammatory parameters in the CSF. To confirm TBE, the presence of TBEV specific antibodies in serum and CSF was evaluated. Patients were treated with Mannitol at a dose of 0.25 g/kg body weight as a 15% solution administered 2–4 times per day. In five patients with meningoencephalitis, Dexamethasone, at a dose of 4–24 mg per day for 4–6 days, was used. The main analgesic used was Acetaminophen.Written informed consent was obtained from all study participants. The study was approved by the Bioethics Committee at the Medical University of Bialystok.CSF samples were collected into polypropylene tubes and venous blood samples were obtained by venipuncture. Venous blood samples were centrifuged to separate the serum. Both CSF and serum samples were aliquoted and frozen at −80 °C until assayed. The samples were collected prior to (sample 1) and following treatment (sample 2). Changes in the tested biomarkers before and after treatment were examined in all patients.κFLC, λFLC, albumin, and IgG in the CSF and serum were measured on the Optilite analyzer (The Binding Site) according to the turbidimetric method. When FLC concentrations were below the limit of detection, we used the corresponding detection limit: CSF κFLC—0.30 mg/L and CSF λFLC—0.65 mg/L.TBEV specific antibodies titer was measured using the Enzygnost Anti-TBE/FSME Virus  Siemens test (OD korr./U).Results were also expressed as κIgG-index, λIgG-index, κFLC-index and λFLC-index. Indexes were calculated according to the following formulas: The κIgG-index and λIgG-index has been developed by Gudowska-Sawczuk et al. whereas the κFLC-index and λFLC-index has been developed by Presslauer S et al. ,14.p < 0.05 as statistically significant.Statistical analysis was performed using Statistica 13.3 . Differences between the tested groups were evaluated by the Mann-Whitney U test. The Spearman rank correlation coefficient was used to measure the degree of association between variables. We considered p = 0.020; p < 0.001; p < 0.001; p < 0.001, respectively).The results of routine laboratory tests in patients with TBE are presented in p = 0.007) while the concentrations of CSF λFLC were markedly elevated after treatment (p = 0.047). The concentrations of serum and CSF κFLC did not differ between sample 1 and sample 2 . The values of serum TBEV IgG. CSF TBEV IgM and CSF TBEV IgG were higher after treatment . The values of serum TBEV IgM were similar in sample 1 and sample 2 (p = 0.449).The results of free light chains concentrations and specific TBEV antibodies, in patients with TBE, are presented in Differences in the κFLC-index, λFLC-index, κIgG-index and λIgG-index values in the tested groups are presented in p = 0.843; p = 0.080; p = 0.293; p = 0.384; p = 0.084; p = 0.849; p = 0.341; p = 0.265, respectively). There were no significant differences in S κFLC, CSF κFLC, S λFLC, CSF λFLC, κFLC-index, λFLC-index, κIgG-index, λIgG-index between patients with meningitis and those with meningoencephalitis following treatment .There were no significant differences in S κFLC, CSF κFLC, S λFLC, CSF λFLC, κFLC-index, λFLC-index, κIgG-index, λIgG-index between patients with meningitis and those with meningoencephalitis prior to treatment  phase of TBE, while the second sample was collected in the recovery phase (disappearance of acute disease symptoms), prior to the patient’s discharge from hospital.In the present study, we observed that serum λFLC concentrations were significantly elevated prior to treatment in comparison to post-treatment values. Furthermore, in sample 1, the levels of serum total IgG were also increased. Therefore, elevated λFLC levels are presumably caused by enhanced production of immunoglobulins during inflammation in patients with TBE. On the other hand, the concentrations of CSF λFLC were elevated following treatment and were almost twice as high as serum λFLC levels.It is well established that serum is the main source of proteins present in the CSF and the protein level is regulated by, inter alia, the permeability of the blood-brain-barrier and the CSF flow rate. Enhanced levels of immunoglobulins in the CSF may indicate increased permeability of BBB or intrathecal synthesis in the CNS. Additionally, FLC exists in two major molecular forms: monomers or dimers. λFLC are often dimers which, in normal conditions, should not pass the blood-brain barrier . TherefoConsequently, in this study, we examined values of four indexes calculated on the basis of λFLC, κFLC, albumin, and IgG concentrations: κFLC-index, λFLC-index, and those developed in our previous study: λIgG-index and κIgG-index . We showThe limitation of the present study was a relatively small sample size and, therefore, future studies should aim to confirm the clinical and prognostic role of free light chains in tick-borne encephalitis in a larger population. The results of our current study confirm that there are differences in serum and CSF concentrations of λ free light chains differ between the initial stage of the disease and following treatment. A decrease in serum λFLC, and an increase in CSF λFLC, after treatment may reflect the intrathecal synthesis of immunoglobulins or the blood-brain barrier disruption.