Blood vessel organoids generated by base editing and harboring single nucleotide variation in Notch3 effectively recapitulate CADASIL-related pathogenesis.

Human blood vessel organoids (hBVOs) offer a promising platform for investigating vascular diseases and identifying therapeutic targets. In this study, we focused on in vitro modeling and therapeutic target finding of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common form of hereditary stroke disorder caused by mutations in the NOTCH3 gene. Despite the identification of these mutations, the underlying pathological mechanism is elusive, and effective therapeutic approaches are lacking. CADASIL primarily affects the blood vessels in the brain, leading to ischemic strokes, migraines, and dementia. By employing CRISPR/Cas9 base-editing technology, we generated human induced pluripotent stem cells (hiPSCs) carrying Notch3 mutations. These mutant hiPSCs were differentiated into hBVOs. The NOTCH3 mutated hBVOs exhibited CADASIL-like pathology, characterized by a reduced vessel diameter and degeneration of mural cells. Furthermore, we observed an accumulation of Notch3 extracellular domain (Notch3ECD), increased apoptosis, and cytoskeletal alterations in the NOTCH3 mutant hBVOs. Notably, treatment with ROCK inhibitors partially restored the disconnection between endothelial cells and mural cells in the mutant hBVOs. These findings shed light on the pathogenesis of CADASIL and highlight the potential of hBVOs for studying and developing therapeutic interventions for this debilitating human vascular disorder.

Neuronal innervation regulates the secretion of neurotrophic myokines and exosomes from skeletal muscle.

Myokines and exosomes, originating from skeletal muscle, are shown to play a significant role in maintaining brain homeostasis. While exercise has been reported to promote muscle secretion, little is known about the effects of neuronal innervation and activity on the yield and molecular composition of biologically active molecules from muscle. As neuromuscular diseases and disabilities associated with denervation impact muscle metabolism, we hypothesize that neuronal innervation and firing may play a pivotal role in regulating secretion activities of skeletal muscles. We examined this hypothesis using an engineered neuromuscular tissue model consisting of skeletal muscles innervated by motor neurons. The innervated muscles displayed elevated expression of mRNAs encoding neurotrophic myokines, such as interleukin-6, brain-derived neurotrophic factor, and FDNC5, as well as the mRNA of peroxisome-proliferator-activated receptor γ coactivator 1α, a key regulator of muscle metabolism. Upon glutamate stimulation, the innervated muscles secreted higher levels of irisin and exosomes containing more diverse neurotrophic microRNAs than neuron-free muscles. Consequently, biological factors secreted by innervated muscles enhanced branching, axonal transport, and, ultimately, spontaneous network activities of primary hippocampal neurons in vitro. Overall, these results reveal the importance of neuronal innervation in modulating muscle-derived factors that promote neuronal function and suggest that the engineered neuromuscular tissue model holds significant promise as a platform for producing neurotrophic molecules.

Particulate Matter Induces Oxidative Stress and Ferroptosis in Human Lung Epithelial Cells.

Numerous toxicological studies have highlighted the association between urban particulate matter (PM) and increased respiratory infections and lung diseases. The adverse impact on the lungs is directly linked to the complex composition of particulate matter, initiating reactive oxygen species (ROS) production and consequent lipid peroxidation. Excessive ROS, particularly within mitochondria, can destroy subcellular organelles through various pathways. In this study, we confirmed the induction of ferroptosis, an iron-dependent cell death, upon exposure to an urban PM using RT-qPCR and signaling pathway analysis. We used KRISS CRM 109-02-004, the certified reference material for the analysis of particulate matter, produced by the Korea Research Institute of Standards and Science (KRISS). To validate that ferroptosis causes lung endothelial toxicity, we assessed intracellular mitochondrial potential, ROS overproduction, lipid peroxidation, and specific ferroptosis biomarkers. Following exposure to the urban PM, a significant increase in ROS generation and a decrease in mitochondrial potential were observed. Furthermore, it induced hallmarks of ferroptosis, including the accumulation of lipid peroxidation, the loss of antioxidant defenses, and cellular iron accumulation. In addition, the occurrence of oxidative stress as a key feature of ferroptosis was confirmed by increased expression levels of specific oxidative stress markers such as NQO1, CYP1B1, FTH1, SOD2, and NRF. Finally, a significant increase in key ferroptosis markers was observed, including xCT/SLC7A11, NQO1, TRIM16, HMOX-1, FTL, FTH1, CYP1B1, CHAC1, and GPX4. This provides evidence that elevated ROS levels induce oxidative stress, which ultimately triggers ferroptosis. In conclusion, our results show that the urban PM, KRISS CRM, induces cellular and mitochondrial ROS production, leading to oxidative stress and subsequent ferroptosis. These results suggest that it may induce ferroptosis through ROS generation and may offer potential strategies for the treatment of lung diseases.

A Spike-like Self-Assembly of Polyaspartamide Integrated with Functionalized Nanoparticles.

The integration of nanoparticles (NPs) into molecular self-assemblies has been extensively studied with the aim of building well-defined, ordered structures which exhibit advanced properties and performances. This study demonstrates a novel strategy for the preparation of a spike-like self-assembly designed to enhance UV blocking. Poly(2-hydroxyethyl aspartamide) (PHEA) substituted with octadecyl chains and menthyl anthranilate (C18-M-PHEA) was successfully synthesized by varying the number of grafted groups to control their morphology and UV absorption. The in situ incorporation of polymerized rod-like TiO2 within the C18-M-PHEA self-aggregates generated spike-like self-assemblies (TiO2@C18-M-PHEA) with a chestnut burr structure in aqueous solution. The results showed that the spike-like self-assemblies integrated with TiO2 NPs exhibited a nine-fold increase in UV protection by simultaneous UV absorption and scattering compared with the pure TiO2 NPs formed via a bulk mixing process. This work provides a novel method for UV protection using self-assembling poly(amino acid)s derivatives integrated with functional nanoparticles to tune their morphology and organization.

Atherosclerotic three-layer nanomatrix vascular sheets for high-throughput therapeutic evaluation.

In vitro atherosclerosis models are essential to evaluate therapeutics before in vivo and clinical studies, but significant limitations remain, such as the lack of three-layer vascular architecture and limited atherosclerotic features. Moreover, no scalable 3D atherosclerosis model is available for making high-throughput assays for therapeutic evaluation. Herein, we report an in vitro 3D three-layer nanomatrix vascular sheet with critical atherosclerosis multi-features (VSA), including endothelial dysfunction, monocyte recruitment, macrophages, extracellular matrix remodeling, smooth muscle cell phenotype transition, inflammatory cytokine secretion, foam cells, and calcification initiation. Notably, we present the creation of high-throughput functional assays with VSAs and the use of these assays for evaluating therapeutics for atherosclerosis treatment. The therapeutics include conventional drugs (statin and sirolimus), candidates for treating atherosclerosis (curcumin and colchicine), and potential gene therapy (miR-146a-loaded liposomes). The high efficiency and flexibility of the scalable VSA functional assays should facilitate drug discovery and development for atherosclerosis.

Comprehensive assessment of the estrogenic activity of resin composites.

Resin-based dental composites have been developed to restore decayed teeth or modify tooth color due to their excellent physical and chemical properties. Such composites may have intrinsic toxicity due to components released into the mouth during the early stage of polymerization, and afterward as a result of erosion or material decomposition. In addition, resin-based dental composites have potential environmental pollutant by elution of monomers and degradation. Since certain monomers of resin matrices are synthesized from bisphenol A (BPA), which acts as an estrogenic endocrine disruptor, these resin matrices may have estrogenic activity. Therefore, the estrogenic endocrine-disrupting activity of various dental composites should be evaluated. In this study, we evaluated the estrogenic endocrine-disrupting activity of 10 resin composites by using a BRET-based estrogen receptor (ER)α and ERß dimerization assays and ER transactivation assay. BPA, BisDMA, BisGMA, BisEMA, TEGDMA, HMBP, and DMPA mediated ERα dimerization, and BPA, BisDMA, and DMPA also mediated ERß dimerization. Except for UDMA and CQ, all the compounds were identified as estrogen agonists or antagonists. In-depth information for the safe use of dental composites was acquired, and it was confirmed how the component of dental composites acts in the ER signaling pathway. Further studies on the low-dose and long-term release of these compounds are needed to ensure the safe use of these resin-based dental composites.

Seven new secondary metabolites isolated from roots of Lespedeza bicolor.

Chemical investigation of a methanol extract obtained from the roots of Lespedeza bicolor identified one new pterocarpene (1), three new pterocarpans (2-4), and three new arylbenzofurans (5-7), and two known compounds (8 and 9). Their structures were determined by interpretations obtained from combined UV, NMR, and HRTOFMS spectroscopic data. Furthermore, the absolute configurations of compounds 2 and 3 were established by the combination of electronic circular dichroism (ECD) calculations and NMR calculations with DP4+ probability analysis. All isolated compounds (1-9) were evaluated for cytotoxicity against the human lung carcinoma cell line A549 and the human hepatoma cell line Huh-7. Compound 4 showed antiproliferative activity against A549 cell line with IC50 value of 24.9 µM. Furthermore, compound 9 exhibited cytotoxicity against Huh-7 cell line with IC50 value of 68.7 µM.

A novel optical coherence tomography-based in vitro method of anti-aging skin analysis using 3D skin wrinkle mimics.

BACKGROUND: Wrinkles represent a characteristic symptom of skin aging. In recent years, various studies have focused on their prevention and/or cure. However, clinical tests are still the only method available to directly detect and evaluate the anti-wrinkle efficacy of various substances. Moreover, no in vitro strategy for such anti-aging skin analysis has been reported. Therefore, in this study, we aimed to develop a novel technology to overcome these limitations. MATERIALS AND METHODS: Full-thickness (FT) skin wrinkle mimics with various widths and depths were fabricated using a collagen stamping method. These were analyzed and compared using 2D and 3D Swept Source-Optical Coherence Tomography (SS-OCT) imaging technologies. RESULTS: SS-OCT demonstrated superficial and cross-sectional images of the wrinkle mimics, and the size of the wrinkles was validated using image analysis. Retinoic acid treatment significantly decreased both the depth and width of wrinkles formed in the FT skin wrinkle mimics. CONCLUSIONS: Using 3D tissue engineering and SS-OCT imaging technologies, we developed a novel in vitro technique that can directly detect skin wrinkles. This significantly efficient method could lead to an alternative strategy for animal experiments and preclinical anti-aging research on the skin.

Surgical treatment of postauricular hidradenitis suppurativa with delayed diagnosis: a case report and literature review.

Hidradenitis suppurativa (HS) is a chronic inflammatory condition that is difficult to diagnose, with a period of 10.0± 9.6 years from symptom onset to diagnosis. A 32-year-old Asian man presented with bilateral postauricular abscesses that first appeared 5 years previously. Despite several incisions and drainage, the symptoms only temporarily improved and continued to recur. On physical examination, chronic scars and sinus tracts were observed around the lesion. Postauricular HS was diagnosed, and surgical treatment was performed. We performed a wide excision and reconstructed the defect using a posterior auricular artery perforator-based keystone flap. Histological examination confirmed the diagnosis of HS. The reconstruction was successful, and there was no recurrence for 2 years after surgery. HS is difficult to diagnose without specific attention. Although the postauricular region is not a typical site of HS, it can occur in this area. Therefore, if a patient presents with recurrent abscesses in the postauricular region, HS should be considered. Additionally, if HS is diagnosed in the postauricular region, wide excision with reconstruction using a posterior auricular artery perforator-based keystone flap can lead to a favorable outcome.

Toenail Paronychium Flap: Novel Surgical Approach for Ingrowing Toenail and Review of the Literature of Conventional Surgical Methods.

Ingrown toenails are most common among school-age children and adolescents though they can be observed at any age. Causes of ingrown toenails are increased curvature, trauma, and external pressure. Treatment of ingrown toenails can be broadly characterized as conservative and surgical. Conservative treatment can be performed using various methods, such as a gutter splint, dental floss, and cotton. Surgical treatments may be divided into two main approaches; narrowing of the nail plate and debulking of periungual tissues. However, these various conservative and surgical treatments have high recurrence rates, and thus, the author used a permanent surgical method based on the use of a paronychium flap to treat a 15-year-old male adolescent with excessive periungual tissues and curved ingrown toenails who did not improve despite conservative and several surgical treatments over 4 years. Subsequently, toenail shape was maintained without recurrence 22 months after surgery, and there were no complaints of inflammation or pain while walking. This simple surgical method can be performed on patients with advanced ingrown toenails due to excessive periungual tissues and nail curvature and can be expected to have permanent effects.

Multi-contrast digital histopathology of mouse organs using quantitative phase imaging and virtual staining.

Quantitative phase imaging (QPI) has emerged as a new digital histopathologic tool as it provides structural information of conventional slide without staining process. It is also capable of imaging biological tissue sections with sub-nanometer sensitivity and classifying them using light scattering properties. Here we extend its capability further by using optical scattering properties as imaging contrast in a wide-field QPI. In our first step towards validation, QPI images of 10 major organs of a wild-type mouse have been obtained followed by H&E-stained images of the corresponding tissue sections. Furthermore, we utilized deep learning model based on generative adversarial network (GAN) architecture for virtual staining of phase delay images to a H&E-equivalent brightfield (BF) image analogues. Using the structural similarity index, we demonstrate similarities between virtually stained and H&E histology images. Whereas the scattering-based maps look rather similar to QPI phase maps in the kidney, the brain images show significant improvement over QPI with clear demarcation of features across all regions. Since our technology provides not only structural information but also unique optical property maps, it could potentially become a fast and contrast-enriched histopathology technique.

Flotillin-1 palmitoylation turnover by APT-1 and ZDHHC-19 promotes cervical cancer progression by suppressing IGF-1 receptor desensitization and proteostasis.

We have shown that insulin-like growth factor-1 (IGF-1) induces palmitoylation turnover of Flotillin-1 (Flot-1) in the plasma membrane (PM) for cell proliferation, after IGF-1 receptor (IGF-1R) signaling activation. However, the enzymes responsible for the turnover have not been identified. Herein, we show that acyl protein thioesterases-1 (APT-1) catalyzes Flot-1 depalmitoylation, and zinc finger DHHC domain-containing protein palmitoyltransferase-19 (ZDHHC-19) repalmitoylation of the depalmitoylated Flot-1 for the turnover in cervical cancer cells. The turnover prevented desensitization of IGF-1R via endocytosis and lysosomal degradation, thereby exerting excessive IGF-1R activation in cervical cancer cells. FLOT1, LYPLA1 and ZDHHC19 were highly expressed, and epithelial-to-mesenchymal transition (EMT)-inducing TIAM1 and GREM1 coordinately upregulated in malignant cervical cancer tissues. And blocking the turnover suppressed the EMT, migration, and invasion of cervical cancer cells. Our study identifies the specific enzymes regulating Flot-1 palmitoylation turnover, and reveals a novel regulatory mechanism of IGF-1-mediated cervical cancer progression.

Deep Learning-Based Glaucoma Screening Using Regional RNFL Thickness in Fundus Photography.

Since glaucoma is a progressive and irreversible optic neuropathy, accurate screening and/or early diagnosis is critical in preventing permanent vision loss. Recently, optical coherence tomography (OCT) has become an accurate diagnostic tool to observe and extract the thickness of the retinal nerve fiber layer (RNFL), which closely reflects the nerve damage caused by glaucoma. However, OCT is less accessible than fundus photography due to higher cost and expertise required for operation. Though widely used, fundus photography is effective for early glaucoma detection only when used by experts with extensive training. Here, we introduce a deep learning-based approach to predict the RNFL thickness around optic disc regions in fundus photography for glaucoma screening. The proposed deep learning model is based on a convolutional neural network (CNN) and utilizes images taken with fundus photography and with RNFL thickness measured with OCT for model training and validation. Using a dataset acquired from normal tension glaucoma (NTG) patients, the trained model can estimate RNFL thicknesses in 12 optic disc regions from fundus photos. Using intuitive thickness labels to identify localized damage of the optic nerve head and then estimating regional RNFL thicknesses from fundus images, we determine that screening for glaucoma could achieve 92% sensitivity and 86.9% specificity. Receiver operating characteristic (ROC) analysis results for specificity of 80% demonstrate that use of the localized mean over superior and inferior regions reaches 90.7% sensitivity, whereas 71.2% sensitivity is reached using the global RNFL thicknesses for specificity at 80%. This demonstrates that the new approach of using regional RNFL thicknesses in fundus images holds good promise as a potential screening technique for early stage of glaucoma.

Maintenance of Hypoimmunogenic Features via Regulation of Endogenous Antigen Processing and Presentation Machinery.

Universally acceptable donor cells have been developed to address the unmet need for immunotypically matched materials for regenerative medicine. Since forced expression of hypoimmunogenic genes represses the immune response, we established universal pluripotent stem cells (PSCs) by replacing endogenous ß2-microglobulin (ß2m) with ß2m directly conjugated to human leukocyte antigen (HLA)-G, thereby simultaneously suppressing HLA-I expression and the natural killer (NK) cell-mediated immune response. These modified human PSCs retained their pluripotency and differentiation capacity; however, surface presentation of HLA-G was absent from subsequently differentiated cells, particularly cells of neural lineages, due to the downregulation of antigen processing and presentation machinery (APM) genes. Induction of APM genes by overexpression of NLR-family CARD domain-containing 5 (NLRC5) or activator subunit of nuclear factor kappa B (NF-κB) heterodimer (RelA) recovered the surface expression of HLA-G and the hypoimmunogenicity of neural cells. Our findings enhance the utility of hypoimmunogenic cells as universal donors and will contribute to the development of off-the-shelf stem-cell therapeutics.

Design, Synthesis and Biological Evaluation of 1,3,5-Triazine Derivatives Targeting hA1 and hA3 Adenosine Receptor.

Adenosine mediates various physiological activities in the body. Adenosine receptors (ARs) are widely expressed in tumors and the tumor microenvironment (TME), and they induce tumor proliferation and suppress immune cell function. There are four types of human adenosine receptor (hARs): hA1, hA2A, hA2B, and hA3. Both hA1 and hA3 AR play an important role in tumor proliferation. We designed and synthesized novel 1,3,5-triazine derivatives through amination and Suzuki coupling, and evaluated them for binding affinities to each hAR subtype. Compounds 9a and 11b showed good binding affinity to both hA1 and hA3 AR, while 9c showed the highest binding affinity to hA1 AR. In this study, we discovered that 9c inhibits cell viability, leading to cell death in lung cancer cell lines. Flow cytometry analysis revealed that 9c caused an increase in intracellular reactive oxygen species (ROS) and a depolarization of the mitochondrial membrane potential. The binding mode of 1,3,5-triazine derivatives to hA1 and hA3 AR were predicted by a molecular docking study.

Highly sensitive pregnancy test kit via oriented antibody conjugation on brush-type ligand-coated quantum beads.

Lateral flow assays (LFA) enable development of portable and rapid diagnostic kits; however, their capacity to detect low levels of disease markers remains poor. Here, we report a highly sensitive pregnancy test kit as a proof of concept, by combining brush-type ligand-coated quantum beads (B-type QBs) and nanobody, which can control the antibody orientation and enhance sensitivity. The brush-type ligand provided excellent dispersion stability and high-binding capacity toward antibody. Fc-binding nanobody increased the antigen-binding capacity of conjugated antibodies on the B-type QBs. To facilitate convenient acquisition of the LFA results, we developed a smartphone-based reader with a 3D-printed optical imaging module, and validated the diagnostic performance of the sensing platform. The pregnancy test kit achieved a 5.1 pg mL-1 limit of detection, corresponding to the levels for early-stage detection of heart disease and malaria. Our LFA application can potentially be expanded to diagnosis other diseases by simply changing the antibody pair in the kit.

Human Blood Vessel Organoids Penetrate Human Cerebral Organoids and Form a Vessel-Like System.

Vascularization of tissues, organoids and organ-on-chip models has been attempted using endothelial cells. However, the cultured endothelial cells lack the capacity to interact with other somatic cell types, which is distinct from developing vascular cells in vivo. Recently, it was demonstrated that blood vessel organoids (BVOs) recreate the structure and functions of developing human blood vessels. However, the tissue-specific adaptability of BVOs had not been assessed in somatic tissues. Herein, we investigated whether BVOs infiltrate human cerebral organoids and form a blood-brain barrier. As a result, vascular cells arising from BVOs penetrated the cerebral organoids and developed a vessel-like architecture composed of CD31+ endothelial tubes coated with SMA+ or PDGFR+ mural cells. Molecular markers of the blood-brain barrier were detected in the vascularized cerebral organoids. We revealed that BVOs can form neural-specific blood-vessel networks that can be maintained for over 50 days.

N-myristoylation regulates insulin-induced phosphorylation and ubiquitination of Caveolin-2 for insulin signaling.

N-myristoylation is a ubiquitous protein lipidation in eukaryotes, but regulatory roles for myristoylation on proteins still remain to be explored. Here, we show that N-myristoylation of Caveolin-2 (Cav-2) controls insulin signaling. Alternative translation initiation (ATI)-yielded truncated form of non-N-myristoylable Cav-2ß and various conditional Cav-2 mutants were compared to full-length form of N-myristoylable Cav-2α. Insulin induced insulin receptor (IR) tyrosine kinase-catalyzed Tyr-19 phosphorylation of N-myristoylable M14A Cav-2 and triggered activation of IR signaling cascade. In contrast, insulin induced ubiquitination of non-N-myristoylable M1A and G2A Cav-2 to facilitate protein-tyrosine phosphatase 1B interaction with IR which desensitized IR signaling through internalization. Metabolic labeling and click chemistry showed palmitoylation of M14A but not M1A and G2A Cav-2. Insulin did not induce phosphorylation of M1A and G2A Cav-2 and Cav-2ß. Like Cav-2α, G2A Cav-2 and Cav-2ß formed large homo-oligomers localized in lipid rafts. These findings show Cav-2 N-myristoylation plays a crucial role to coordinate its phosphorylation, palmitoylation, and ubiquitination to control insulin signaling.

The synergistic effect of biomimetic electrical stimulation and extracellular-matrix-mimetic nanopattern for upregulating cell activities.

Endogenous bioelectric signaling and the extracellular matrix (ECM) are factors that have a great effect on the performance of cellular functions. Presenting an experimental platform to confirm the synergy effects of an electrical stimulation, which simulates endogenous bioelectricity, and nanopatterns that can be precisely fabricated in various patterns sizes makes it possible to consider those factors effectively. Herein, we have performed a comparison of cellular response to each of general electrical stimulation and biomimetic electrical stimulation (BES) and demonstrated the synergy effects of electrical stimulation and ECM-mimetic nanopatterns. BES has provided the most remarkable proliferation among different types of electrical stimulation and upregulated the behavior of cells through synergy effects with ECM-mimetic nanopatterns. Thus, it is believed that using the synergy effects of BES and ECM-like nanopatterns has broad applications in the biomedical field, such as cell culture with electrical stimulation, induction of cell growth, tissue repair, etc.

Which pathologic staining method can visualize the hyperacute infarction lesion identified by diffusion MRI?: A comparative experimental study.

BACKGROUND: The study aimed to establish a staining method that could delineate the macroscopic lesion boundary of a hyperacute infarction depicted by diffusion-weighted MRI (DWI) and to validate the infarction boundary by comparing different staining methods. NEW METHOD: Thirteen rats with 1 -h middle cerebral artery (MCA) infarction were included. Five different staining methods (Hematoxylin and eosin (H&E), Nissl, 2,3,5-triphenyltetrazolium hydrochloride (TTC), microtubule associated protein 2 (MAP2), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stains) were used to identify whether the hyperacute infarction could be histopathologically identified. Dice indices were compared to evaluate similarities in the lesion area ascertained by DWI and the staining methods. Through macroscopic lesion delineation, each region was subdivided into abnormal regions in all three stains (ROIA), abnormal in two stains (ROIB), and abnormal in only one (ROIC). Microscopic cellular changes were evaluated and graded according to each region. RESULTS: Mean Dice indices of the H&E stain were significantly higher than those of the Nissl- and MAP2-stained specimens (0.83 ±â€¯0.052, 0.58 ±â€¯0.107, and 0.56 ±â€¯0.059, respectively; p = 0.000). Grading scores for ROIs in the DWI abnormal lesions varied by region: ROIA exhibited the most severe damage [median (IQR), 3 (1)], followed respectively by ROIB [median (IQR), 2 (0)] and ROIC [median (IQR), 1 (0)] COMPARISON WITH EXISTING METHODS: H&E stain best reflects 1 h hyperacute DWI abnormal lesions. CONCLUSIONS: H&E stain allowed for the macroscopic delineation of the 1 h DWI-abnormal lesions, while MAP2 and Nissl stains could only partially depict lesions.