Computer-Aided Multiphoton Microscopy Diagnosis of 5 Different Primary Architecture Subtypes of Meningiomas.

Meningiomas rank among the most common intracranial tumors, and surgery stands as the primary treatment modality for meningiomas. The precise subtyping and diagnosis of meningiomas, both before and during surgery, play a pivotal role in enabling neurosurgeons choose the optimal surgical program. In this study, we utilized multiphoton microscopy (MPM) based on 2-photon excited fluorescence and second-harmonic generation to identify 5 common meningioma subtypes. The morphological features of these subtypes were depicted using the MPM multichannel mode. Additionally, we developed 2 distinct programs to quantify collagen content and blood vessel density. Furthermore, the lambda mode of the MPM characterized architectural and spectral features, from which 3 quantitative indicators were extracted. Moreover, we employed machine learning to differentiate meningioma subtypes automatically, achieving high classification accuracy. These findings demonstrate the potential of MPM as a noninvasive diagnostic tool for meningioma subtyping and diagnosis, offering improved accuracy and resolution compared with traditional methods.

Double-modified oncolytic adenovirus armed with a recombinant interferon-like gene enhanced abscopal effects against malignant glioma.

Background: The development of new therapies for malignant gliomas has been stagnant for decades. Through the promising outcomes in clinical trials of oncolytic virotherapy, there is now a glimmer of hope in addressing this situation. To further enhance the antitumor immune response of oncolytic viruses, we have equipped a modified oncolytic adenovirus (oAds) with a recombinant interferon-like gene (YSCH-01) and conducted a comprehensive evaluation of the safety and efficacy of this modification compared to existing treatments. Methods: To assess the safety of YSCH-01, we administered the oAds intracranially to Syrian hamsters, which are susceptible to adenovirus. The efficacy of YSCH-01 in targeting glioma was evaluated through in vitro and in vivo experiments utilizing various human glioma cell lines. Furthermore, we employed a patient-derived xenograft model of recurrent glioblastoma to test the effectiveness of YSCH-01 against temozolomide. Results: By modifying the E1A and adding survivin promoter, the oAds have demonstrated remarkable safety and an impressive ability to selectively target tumor cells. In animal models, YSCH-01 exhibited potent therapeutic efficacy, particularly in terms of its distant effects. Additionally, YSCH-01 remains effective in inhibiting the recurrent GBM patient-derived xenograft model. Conclusions: Our initial findings confirm that a double-modified oncolytic adenovirus armed with a recombinant interferon-like gene is both safe and effective in the treatment of malignant glioma. Furthermore, when utilized in combination with a targeted therapy gene strategy, these oAds exhibit a more profound effect in tumor therapy and an enhanced ability to inhibit tumor growth at remote sites.

Ruptured Anterior Communicating Artery Aneurysms Concomitant with Small Intracranial Hematoma: Evacuating Hematoma or Not?

Objective: To identify whether intracranial hematoma (ICH) evacuation improves the prognosis of patients with ruptured anterior communicating artery (AcomA) aneurysms concomitant with small ICHs (≥10 mL and <25 mL). Methods: Data on patients diagnosed with small ICHs secondary to ruptured AcomA aneurysms who underwent surgery in our department between January 2010 and February 2018 was retrospectively analyzed. The patients were divided into two groups based on whether the hematoma was evacuated. The modified Rankin Scale (mRS) was used to assess prognosis six months after onset. Results: The study recruited 58 patients, 19 of whom underwent aneurysm clipping and ICH evacuation. While 33 patients underwent aneurysm clipping, 6 patients underwent coiling embolism without ICH evacuation. The average ICH volume was 15.27±4.07 mL. In the hematoma-evacuated group, 13 (68.4%) patients had unfavorable outcomes (mRS scores of 4 to 6). In the non-evacuated hematoma group, 13 (33.3%) patients had unfavorable outcomes (P = 0.001), postoperative infarction occurred in 11 (57.9%) patients in the hematoma evacuation group and 9 (23.1%) patients in the other group (P = 0.009). Conclusion: ICH evacuation was associated with unfavorable outcomes and postoperative infarction in ruptured AcomA aneurysms with concomitant small hematomas (<25 mL). Aneurysm clipping or coiling without ICH evacuation may be a safe and effective choice; however, further investigation is needed.

Automatic and label-free detection of meningioma in dura mater using the combination of multiphoton microscopy and image analysis.

Significance: To prevent meningioma recurrence, it is necessary to detect and remove all corresponding tumors intraoperatively, including those in the adjacent dura mater. Aim: Currently, the removal of meningiomas from the dura mater depends solely on cautious visual identification of lesions by a neurosurgeon. Inspired by the requirements for resection, we propose multiphoton microscopy (MPM) based on two-photon-excited fluorescence and second-harmonic generation as a histopathological diagnostic paradigm to assist neurosurgeons in achieving precise and complete resection. Approach: Seven fresh normal human dura mater samples and 10 meningioma-infiltrated dura mater samples, collected from 10 patients with meningioma, were acquired for this study. First, multi-channel mode and lambda mode detection were utilized in the MPM to characterize the architectural and spectral features of normal and meningioma-infiltrated dura mater, respectively. Three imaging algorithms were then employed to quantify the architectural differences between the normal and meningioma-infiltrated dura mater through calculations of the collagen content, orientation, and alignment. Finally, MPM was combined with another custom-developed imaging algorithm to locate the meningioma within the dura mater and further delineate the tumor boundary. Results: MPM not only detected meningioma cells in the dura mater but also revealed the morphological and spectral differences between normal and meningioma-infiltrated dura mater, providing quantitative information. Furthermore, combined with a self-developed image-processing algorithm, the precise borders of meningiomas in the dura mater could be accurately delineated. Conclusions: MPM can automatically detect meningiomas in the dura mater label-free. With the development of advanced multiphoton endoscopy, MPM combined with image analysis can provide decision-making support for histopathological diagnosis, as well as offer neurosurgeons more precise intraoperative resection guidance for meningiomas.

Global, regional, and national burden and attributable risk factors of neurological disorders: The Global Burden of Disease study 1990-2019.

Background: Neurological disorders are a major and increasing global health challenge, which accounts for a substantial portion of the disease burden worldwide. The aim of this systematic analysis is to present the most comprehensive and up-to-date estimates of disease burden, epidemiological trends, and attributable risk factors of neurological disorders at global, regional, and national levels. Methods: We extracted data of 18 neurological disorders from the Global Burden of Disease 2019 study database. The burden of neurological disorders was measured using the incidence, prevalence, mortality, and disability-adjusted life years (DALYs), and further described according to age, sex, year, geographical location and socio-demographic Index (SDI). All estimates were presented with corresponding 95% uncertainty intervals (UIs). Findings: Globally, in 2019, there were nearly 10 million deaths and 349 million DALYs due to neurological disorders. Among the 18 neurological disorders, stroke was the biggest contributor to DALYs (143232.18 [95%UI 133095.81-153241.82] in thousands) and deaths (6552.72 [95%UI 5995.20-7015.14] in thousands), followed by neonatal encephalopathy due to birth asphyxia and trauma. From 1990 to 2019, the DALYs of neurological diseases belonging to the communicable, maternal, neonatal and nutritional categories showed a sharp decrease, while Alzheimer's disease and other dementias and Parkinson's disease showed a large increase. Neurological disorders exhibited different profiles in different regions and age groups. A significant correlation between the SDI and the age-standardized DALY rates was also found except for Alzheimer's disease and other dementias. In addition, risk factors such as high systolic blood pressure, low birth weight and short gestation period, and metabolic risk contribute significantly to neurological disorders. Interpretation: The overall burden of neurological disorders has increased from 1990 to 2019, especially for non-communicable neurological disorders. The substantial variations of burden across regions emphasize the need for region-specific interventional strategies and allocation of resources based on priorities.

Integrative and comparative single-cell analysis reveals transcriptomic difference between human tumefactive demyelinating lesion and glioma.

Tumefactive demyelinating lesion (TDL) is an immune-mediated disease which can be misdiagnosed as glioma. At present, there is no study comparing difference between the two disorders at the cellular level. Here, we perform integrative and comparative single-cell RNA sequencing (ScRNA-seq) transcriptomic analysis on TDL and glioma lesions. At single-cell resolution, TDL is comprised primarily of immune cells, which is completely different from glioma. The integrated analysis reveals a TDL-specific microglial subset involving in B cell activation and proliferation. Comparative analysis highlights remyelination function of glial cells and demyelination function of T cells in TDL. Subclustering and pseudotime trajectory analysis of T cells in TDL reveal their heterogeneity and diverse functions involving in TDL pathogenesis and recovery process. Our study identifies substantial differences between TDL and glioma at single-cell resolution. The observed heterogeneity and potentially diverse functions of cells in TDL may be critical in disease progression.

Single-cell RNA sequencing reveals intra-tumoral heterogeneity of glioblastoma and a pro-tumor subset of tumor-associated macrophages characterized by EZH2 overexpression.

BACKGROUND: Glioblastoma (GBM) is a highly heterogeneous disease with poor clinical outcome. AIM: To comprehensively dissect molecular landscape of GBM and heterogeneous distribution and potential role of Enhancer of zeste homolog 2 (EZH2) in tumor microenvironment (TME). METHODS: Single-cell RNA sequencing (scRNA-seq) analysis was performed in GBM samples from 8 patients. Deconvolution analysis, immunofluorescence (IF) microscopy, reverse-transcription quantitative polymerase chain reaction (RT-qPCR), colony formation experiments, and Cell Counting Kit-8 (CCK-8) assays were performed to confirmed the potential role of EZH2 in TME cells. RESULTS: Malignant cells exhibited remarkable heterogeneity in abnormal metabolic patterns. A mesenchymal-2-like (MES2-like) GBM subcluster with glial-immune dual feature was firstly discovered, which were associated with highly activated hallmark pathways, immune evasion associated transcription factor (IRF8), and poor survival. The oncogene, EZH2, was heterogeneously expressed in malignant cells and immune cells consistent with proliferative genes, cell-cycle transcription factors, and similar activated hallmark pathways. In a tumor-associated macrophages (TAMs) subset (macrophage.3), EZH2 was highly expressed with similar changes of transcriptomic dynamics with cell-cycle genes and macrophages M2-phetotype genes. In addition, the subset tightly interacted with malignant cells. Deconvolution analysis showed increased abundance of the subset in GBM compared to low-grade glioma (LGG) and significant association with worse prognosis. Functional verification experiments confirmed the pro-tumor role of TAMs with EZH2 overexpression in GBM. CONCLUSIONS: Our study illustrated a MES2-like GBM subcluster characterized by glial-immune dual feature and highlighted the pro-tumor role of a TAMs subset characterized by EZH2 overexpression.

Warburg effect-promoted exosomal circ_0072083 releasing up-regulates NANGO expression through multiple pathways and enhances temozolomide resistance in glioma.

BACKGROUND: Temozolomide (TMZ) resistance limits its application in glioma. Exosome can carry circular RNAs (circRNAs) to regulate drug resistance via sponging microRNAs (miRNAs). miRNAs can control mRNA expression by regulate the interaction with 3'UTR and methylation. Nanog homeobox (NANOG) is an important biomarker for TMZ resistance. Hitherto, it is unknown about the role of exosomal hsa_circ_0072083 (circ_0072083) in TMZ resistance in glioma, and whether it is associated with NANOG via regulating miRNA sponge and methylation. METHODS: TMZ-resistant (n = 36) and sensitive (n = 33) patients were recruited. The sensitive cells and constructed resistant cells were cultured and exposed to TMZ. circ_0072083, miR-1252-5p, AlkB homolog H5 (ALKBH5) and NANOG levels were examined via quantitative reverse transcription polymerase chain reaction and western blot. The half maximal inhibitory concentration (IC50) of TMZ, cell proliferation, apoptosis, migration and invasion were analyzed via Cell Counting Kit-8, colony formation, flow cytometry, wound healing and transwell assays. The in vivo function was assessed using xenograft model. The N6-methyladenosine (m6A) level was analyzed via methylated RNA immunoprecipitation (MeRIP). Target relationship was investigated via dual-luciferase reporter assay and RNA immunoprecipitation. Warburg effect was investigated via lactate production, glucose uptake and key enzymes expression. Exosome was isolated and confirmed via transmission electron microscopy and specific protein expression. RESULTS: circ_0072083 expression was increased in TMZ-resistant glioma tissues and cells. circ_0072083 knockdown restrained the resistance of resistant cells via decreasing IC50 of TMZ, proliferation, migration, invasion and xenograft tumor growth and increasing apoptosis. circ_0072083 silence reduced NANOG expression via blocking ALKBH5-mediated demethylation. circ_0072083 could regulate NANOG and ALKBH5 via targeting miR-1252-5p to control TMZ resistance. Warburg effect promoted the release of exosomal circ_0072083 in resistant cells. Exosomal circ_0072083 from resistant cells increased the resistance of sensitive cells to TMZ in vitro and xenograft model. Exosomal circ_0072083 level was enhanced in resistant patients, and it had a diagnostic value and indicated a lower overall survival in glioma. CONCLUSION: Exosomal circ_0072083 promoted TMZ resistance via increasing NANOG via regulating miR-1252-5p-mediated degradation and demethylation in glioma.

Glioblastoma in a female neurofibromatosis 1 patient without IDH1, BRAF V600E, and TERT promoter mutations: A case report.

RATIONALE: Glioblastoma is the most lethal and common malignant brain tumor but rare in patients with neurofibromatosis type 1. The clinical findings and pathological findings with gene signatures in female patients have not been well clarified. PATIENT CONCERNS: A 51-year-old female patient complained of headache and left limb weakness lasting for 20 days. The patient underwent a cesarean section 20 years ago and hysterectomy 1 year ago because of uterine leiomyomas. Multiple café-au-lait spots and neurofibromas were found over patient's chest, neck, back, and arms. The myodynamia of left distant and proximate epipodite were grade 0 and grade 1 respectively. The myodynamia of lower left limb was grade 3. DIAGNOSES: Magnetic resonance imaging revealed a malignant lesion which was most likely a glioblastoma in the right temporo-parietal lobe, approximately 5.6 × 5.9 × 6.9 cm in size with a rounded boundary. INTERVENTIONS: A right temporo-parietal craniotomy was performed to resect the space-occupying lesion for gross total removal. Then, the patient received concurrent chemoradiotherapy. Histological examination confirmed a glioblastoma without v-RAF murine sarcoma viral oncogene homolog B1 gene, isocitrate dehydrogenase 1 gene, and telomerase reverse transcriptase gene promoter mutations. OUTCOMES: After surgery, the headache was relieved and the muscular strength of left limbs did improve. After receiving the standard treatment regimen, the patient was alive at 13 months follow-up. LESSONS: This is the first reported glioblastoma in female neurofibromatosis type 1 patient without v-RAF murine sarcoma viral oncogene homolog B1 gene, isocitrate dehydrogenase 1 gene, and telomerase reverse transcriptase gene promoter mutations. Tumors in adult patients with these signatures were less aggressive with well-circumscribed border and had long-term survivals which strengthened the evidence that these patients may comprise a unique subset in glioblastoma.

Serum Inflammatory Biomarkers Contribute to the Prognosis Prediction in High-Grade Glioma.

BACKGROUND: To evaluate the prognostic value of serum inflammatory biomarkers and develop a risk stratification model for high-grade glioma (HGG) patients based on clinical, laboratory, radiological, and pathological factors. MATERIALS AND METHODS: A retrospective study of 199 patients with HGG was conducted. Patients were divided into a training cohort (n = 120) and a validation cohort (n = 79). The effects of potential associated factors on the overall survival (OS) time were investigated and the benefits of serum inflammatory biomarkers in improving predictive performance was assessed. Univariable and multivariable Cox regression analyses, the least absolute shrinkage and selection operator (LASSO) regression analysis, and support vector machines (SVM) were used to select variables for the final nomogram model. RESULTS: After multivariable Cox, LASSO, and SVM analysis, in addition to 3 other clinico-pathologic factors, platelet-to-lymphocyte ratio (PLR) >144.4 (hazard ratio [HR], 2.05; 95% confidence interval [CI], 1.25-3.38; P = 0.005) were left for constructing the predictive model. The model with PLR exhibited a better predictive performance than that without them in both cohorts. The nomogram based on the model showed an excellent ability of discrimination in the entire cohort (C-index, 0.747; 95%CI, 0.706-0.788). The calibration curves showed good consistency between the predicted and observed survival probability. CONCLUSION: Our study confirmed the prognostic value of serum inflammatory biomarkers including PLR and established a comprehensive scoring system for the OS prediction in HGG patients.

Exosome-mediated transfer of circRNA CircNFIX enhances temozolomide resistance in glioma.

Development of chemotherapy resistance remains a major obstacle for glioma management. Exosome-mediated transfer of circular RNAs (circRNAs) are being found to have relevance to many human cancers, including glioma. The purpose of this study is to explore the effect and underlying mechanism of exosomal circRNA nuclear factor I X (CircNFIX) on temozolomide (TMZ) chemoresistance in glioma. Our results indicated that exosomal CircNFIX was up-regulated in the serum of TMZ-resistant patients and predicted poor prognosis. Exosomal CircNFIX from TMZ-resistant cells conferred TMZ resistance to recipient sensitive cells through the enhancement of cell migration and invasion and the repression of cell apoptosis under TMZ exposure. CircNFIX directly interacted with miR-132 by binding to miR-132. CircNFIX knockdown enhanced TMZ sensitivity in resistant glioma cells by up-regulating miR-132. Additionally, exosomal CircNFIX promoted tumor growth and its depletion enhanced TMZ sensitivity in glioma cells in vivo. Taken together, our study suggests that exosome-mediated transfer of CircNFIX enhances TMZ resistance in glioma at least partially through sponging miR-132, highlighting a potentially prognostic biomarker and therapeutic target for improving the clinical benefits of TMZ treatment in patients with glioma.

Rapid, label-free detection of intracranial germinoma using multiphoton microscopy.

Accurate histopathological diagnosis is essential for facilitating the optimal surgical management of intracranial germinoma. Current intraoperative histological methods are time- and labor-intensive and often produce artifacts. Multiphoton microscopy (MPM) is a label-free imaging technique that can produce intraoperative histological images of fresh, unprocessed surgical specimens. We employ an MPM based on second-harmonic generation and two-photon excited fluorescence microscopy to image fresh, unfixed, and unstained human germinoma specimens. We show that label-free MPM is not only capable of identifying various cells in human germinoma tissue but also capable of revealing the characteristics of germinoma such as granuloma, stromal fibrosis, calcification, as well as the abnormal and uneven structures of blood vessels. In conjunction with custom-developed image-processing algorithms, MPM can further quantify and characterize the extent of stromal fibrosis and calcification. Our results provide insight into how MPM can deliver rapid diagnostic histological data that could inform the surgical management of intracranial germinoma.

Quantitative assessment of microenvironment characteristics and metabolic activity in glioma via multiphoton microscopy.

Tumor microenvironment and metabolic activity in gliomas are the important biomarkers to evaluate the progression of gliomas. Many evidences have suggested that the targeting of metabolic activity and tumor microenvironment simultaneously can be more effective to take the tumor therapy. Therefore, the noninvasive, accurate assessment of tumor microenvironment and metabolic activity is quite important in clinical practice. Multiphoton microscopy (MPM), based on two-photon-excited fluorescence and second harmonic generation was performed on unstained glioma tissues. With our combined image analysis approaches, our research findings indicate that MPM is able to qualitatively and quantitatively describe the microenvironment characteristics in gliomas, such as collage deposition in extracellular matrix, lymphocyte infiltration and tumor angiogenesis, etc. Meanwhile, the metabolic activity can also be quantitatively evaluated by optical redox ratio, NADH and FAD intensity. With the microendoscope and fiberscope are portable, MPM technique can be used to perform in-vivo studies and clinical examinations in gliomas.

Automatic and label-free identification of blood vessels in gliomas using the combination of multiphoton microscopy and image analysis.

Currently, the targeted treatment of tumor based on the tumor microenvironment is newly developed. Blood vessels are the key parts in the tumor microenvironment, which is taken as a new visible target for tumor therapy. Multiphoton microscopy (MPM), based on the second harmonic generation and two-photon excited fluorescence, is available to make the label-free analysis on the blood vessels in human gliomas. MPM can reveal the vascular morphological characteristics in gliomas, including vascular malformation, intense vascular proliferation, perivascular collagen deposition, perivascular lymphocytes aggregation and microvascular proliferation. In addition, the image analysis algorithms were developed to automatically calculate the perivascular collagen content, vascular cavity area, lumen area, wall area and vessel number. Thus, the vascular morphology, the perivascular collagen deposition and intense vascular proliferation degree can be further quantitatively characterized. Compared with the pathological analysis, the combination of MPM and image analysis has potential advantages in making a quantitative and qualitative analyzing on vascular morphology in glioma microenvironment. As micro-endoscope and two-photon fiberscope are technologically improved, this combined method will be a useful imaging way to make the real-time research on the targeting tumor microenvironment in gliomas.

CircNFIX promotes progression of glioma through regulating miR-378e/RPN2 axis.

BACKGROUND: Circular RNA nuclear factor I X (circNFIX) has been reported to play an important role in glioma progression. However, the mechanism by which circNFIX participates in glioma progression remains poorly understood. METHODS: GERIA online were used to analyze the abnormally expressed genes in glioma tissues. The expression levels of circNFIX, microRNA (miR)-378e and Ribophorin-II (RPN2) were measured by quantitative real-time polymerase chain reaction or western blot. Cell cycle distribution, apoptosis, glycolysis, migration and invasion were determined by flow cytometry, special kit and trans-well assays, respectively. The target association between miR-378e and circNFIX or RPN2 was confirmed by luciferase reporter assay, RNA immunoprecipitation and pull-down. Xenograft model was established to investigate the role of circNFIX in vivo. RESULTS: The expression of circNFIX was enhanced in glioma tissues and cells compared with matched controls and high expression of circNFIX indicated poor outcomes of patients. Knockdown of circNFIX led to arrest of cell cycle, inhibition of glycolysis, migration and invasion and promotion of apoptosis in glioma cells. circNFIX was a sponge of miR-378e. miR-378e overexpression suppressed cell cycle process, glycolysis, migration and invasion but promoted apoptosis. miR-378e silence abated the suppressive role of circNFIX knockdown in glioma progression. RPN2 as a target of miR-378e was positively regulated via circNFIX by competitively sponging miR-378e. Silencing circNFIX decreased glioma xenograft tumor growth by regulating miR-378e/RPN2 axis. CONCLUSION: Knockdown of circNFIX inhibits progression of glioma in vitro and in vivo by increasing miR-378e and decreasing RPN2, providing a novel mechanism for understanding the pathogenesis of glioma.

Ischemia changes and tolerance ratio of evoked potential monitoring in intracranial aneurysm surgery.

OBJECTIVE: We assessed the relationship between cerebral ischemia-induced changes in evoked potentials and the degree of ischemia tolerance. METHODS: 47 patients underwent somatosensory evoked potential (SEP) and motor evoked potential (MEP) monitoring in intracranial aneurysm surgery. Three duration parameters (time) were recorded: Time 1, from the starting of temporary occlusion unavoidable in aneurysm surgery to the time the evoked potentials decrease from basal level to reaching the warning criterion; Time 2, from evoked potentials reaching the warning criterion to the time the blood flow was resumed; Time 3, after resuming the blood flow, the time it took the evoked potentials to recover to baseline. All three times can be reliably calculated in the SEP recording, but not in the MEP recording which consisted of either unchanged amplitudes or abruptly changing amplitudes, making it impossible to obtain Time 1. The ischemic tolerance ratio (ITR) was calculated as ITR=time 2/time 1×100%. New decreasing myodynamia and fresh infarction after the surgery were employed for evaluating neurological deficits postoperatively, and their correlations with the ischemia-induced changes of evoked potentials recorded during the surgery were analyzed. RESULTS: We found a change in SEPs in 12 patients whose cerebral ischemia was induced by temporary occlusion of the aneurysm's parent artery. We also found the development of postoperative neurological deficits in 4 patients whose ischemic tolerance ratio (ITR) reached over 80%, while no deficits were found in the other 8 patients whose ITR was less than 50%. MEP changes were seen in 4 patients whose cerebral ischemia was caused by accidentally clamping the perforating branches, causing the development of postoperative neurological deficits but not necessarily leading to significant SEP changes. CONCLUSION: The Ischemia tolerance ratio (ITR) in SEP recordings is valuable to predicting postoperative neurological deficits caused by temporary occlusion of aneurysm's parent artery. Maintaining the ITR under 50% during operation can effectively avoid postoperative neurological deficits, while an ITR above 80% reliably forecasts postoperative neurological deficits. Complementary to SEPs, MEP recordings are particularly valuable in monitoring ischemic effects caused by accidentally clamping perforating branches. Taken together, this system of monitoring makes it possible to promptly adjust surgery procedures and minimize postoperative neurological deficits.