Histological characterization and development of mesial surface sulci in the human brain at 13-15 gestational weeks through high-resolution histology.

Cellular-level anatomical data from early fetal brain are sparse yet critical to the understanding of neurodevelopmental disorders. We characterize the organization of the human cerebral cortex between 13 and 15 gestational weeks using high-resolution whole-brain histological data sets complimented with multimodal imaging. We observed the heretofore underrecognized, reproducible presence of infolds on the mesial surface of the cerebral hemispheres. Of note at this stage, when most of the cerebrum is occupied by lateral ventricles and the corpus callosum is incompletely developed, we postulate that these mesial infolds represent the primordial stage of cingulate, callosal, and calcarine sulci, features of mesial cortical development. Our observations are based on the multimodal approach and further include histological three-dimensional reconstruction that highlights the importance of the plane of sectioning. We describe the laminar organization of the developing cortical mantle, including these infolds from the marginal to ventricular zone, with Nissl, hematoxylin and eosin, and glial fibrillary acidic protein (GFAP) immunohistochemistry. Despite the absence of major sulci on the dorsal surface, the boundaries among the orbital, frontal, parietal, and occipital cortex were very well demarcated, primarily by the cytoarchitecture differences in the organization of the subplate (SP) and intermediate zone (IZ) in these locations. The parietal region has the thickest cortical plate (CP), SP, and IZ, whereas the orbital region shows the thinnest CP and reveals an extra cell-sparse layer above the bilaminar SP. The subcortical structures show intensely GFAP-immunolabeled soma, absent in the cerebral mantle. Our findings establish a normative neurodevelopment baseline at the early stage.

Systematic development of immunohistochemistry protocol for large cryosections-specific to non-perfused fetal brain.

BACKGROUND: Immunohistochemistry (IHC) is an important technique in understanding the expression of neurochemical molecules in the developing human brain. Despite its routine application in the research and clinical setup, the IHC protocol specific for soft fragile fetal brains that are fixed using the non-perfusion method is still limited in studying the whole brain. NEW METHOD: This study shows that the IHC protocols, using a chromogenic detection system, used in animals and adult humans are not optimal in the fetal brains. We have optimized key steps from Antigen retrieval (AR) to chromogen visualization for formalin-fixed whole-brain cryosections (20 µm) mounted on glass slides. RESULTS: We show the results from six validated, commonly used antibodies to study the fetal brain. We achieved optimal antigen retrieval with 0.1 M Boric Acid, pH 9.0 at 70°C for 20 minutes. We also present the optimal incubation duration and temperature for protein blocking and the primary antibody that results in specific antigen labeling with minimal tissue damage. COMPARISON WITH EXISTING METHODS: The IHC protocol commonly used for adult human and animal brains results in significant tissue damage in the fetal brains with little or suboptimal antigen expression. Our new method with important modifications including the temperature, duration, and choice of the alkaline buffer for AR addresses these pitfalls and provides high-quality results. CONCLUSION: The optimized IHC protocol for the developing human brain (13-22 GW) provides a high-quality, repeatable, and reliable method for studying chemoarchitecture in neurotypical and pathological conditions across different gestational ages.

A technology platform for standardized cryoprotection and freezing of large-volume brain tissues for high-resolution histology.

Understanding and mapping the human connectome is a long-standing endeavor of neuroscience, yet the significant challenges associated with the large size of the human brain during cryosectioning remain unsolved. While smaller brains, such as rodents and marmosets, have been the focus of previous connectomics projects, the processing of the larger human brain requires significant technological advancements. This study addresses the problem of freezing large brains in aligned neuroanatomical coordinates with minimal tissue damage, facilitating large-scale distortion-free cryosectioning. We report the most effective and stable freezing technique utilizing an appropriate choice of cryoprotection and leveraging engineering tools such as brain master patterns, custom-designed molds, and a continuous temperature monitoring system. This standardized approach to freezing enables high-quality, distortion-free histology, allowing researchers worldwide to explore the complexities of the human brain at a cellular level. Our approach combines neuroscience and engineering technologies to address this long-standing challenge with limited resources, enhancing accessibility of large-scale scientific endeavors beyond developed countries, promoting diverse approaches, and fostering collaborations.

Structural and functional changes among diabetics with no diabetic retinopathy and mild non-proliferative diabetic retinopathy using swept-source optical coherence tomography angiography and photopic negative response.

PURPOSE: To assess the structural and functional changes among diabetics with no diabetic retinopathy (NDR) and mild non-proliferative diabetic retinopathy (NPDR) using swept-source optical coherence tomography angiography (SSOCTA) and photopic negative response (PhNR) and to find the earliest changes. METHODS: This was a prospective, cross-sectional, case-control study. Participants with minimum 5 years of diabetes mellitus (DM) were recruited and classified as NDR and mild NPDR based on fundus findings. Age-matched normals with nil ocular pathology were considered as controls. SSOCTA scan acquisition (6*6 mm angiography), followed by full field photopic electroretinography (FFERG) and red on blue PhNR (R/B PhNR) were done with complete pupillary dilatation. RESULTS: A total of 88 participants were included with 35 controls, 39 NDR and 14 mild NPDR subjects. Vessel density of the superficial capillary plexus (SCP) and deep capillary plexus (DCP) of mild NPDR were significantly reduced compared to the controls (17.12 ± 2.65 mm-1 vs. 18.75 ± 0.90 mm-1, p = 0.025 and 7.96 ± 3.92 mm-1 vs. 11.83 ± 3.05 mm-1, p = 0.001 respectively). None of the parameters of controls had significant difference compared to NDR group (p > 0.05). The amplitudes of white on white (W/W) a-wave, W/W b-wave, red on blue (R/B) PhNR baseline to trough (BT) and R/B PhNR peak to trough in controls were significantly high compared to NDR and mild NPDR. Amplitude of R/B PhNR BT had the maximum area under the curve of 75.9% with a sensitivity and specificity of 94.3and 77.4%, respectively. CONCLUSION: A significant decrease in functional changes as measured by ERG especially PhNR, is seen even among the NDR group compared to controls unlike SSOCTA parameters that measures very early vascular structural changes. PhNR is a sensitive test to identify early preclinical changes in DR when microvascular structural changes as determined by SSOCTA are normal.

Comparison of broadband and monochromatic photopic negative response in eyes of patients with diabetes with no diabetic retinopathy and different stages of diabetic retinopathy.

PURPOSE: To evaluate the change in broadband (W/W), red on blue (R/B), and blue on yellow (B/Y) photopic negative response (PhNR) in patients with diabetes mellitus with no diabetic retinopathy (no DR) and different stages of DR and compare it with age-matched controls. This study was performed to provide a single PhNR protocol that can be used for early diagnosis of DR. METHODS: It was a cross-sectional case-control study done in a hospital setup. Patients with diabetes with no DR and different stages of DR with no other associated ocular pathologies were included. Age-matched controls with no retinal pathologies were also included for comparison. All subjects underwent detailed ophthalmic examination and W/W, R/B, and B/Y electroretinography. Fifty control eyes and 52 treatment naïve eyes of 52 patients with diabetes [no DR = 11, mild nonproliferative diabetic retinopathy (NPDR) =11, moderate NPDR = 10, severe NPDR = 9, and proliferative DR = 11] were included in the study. RESULTS: On comparing the ERG responses in patients with diabetes and age-matched controls, a significant reduction (P < 0.05) was noted in the amplitudes of a-wave (39.78 ± 11.34 µV vs. 67.28 ± 12.88 µV), b-wave (116.25 ± 45.25 vs. 134.39 ± 28.78 µV), W/W PhNR (33.86 ± 17.33 vs. 67.18 ± 15.99 µV), R/B PhNR (28.77 ± 15.85 vs. 53.48 ± 14.15 µV), and B/Y PhNR (55.04 ± 32.63 vs. 104.79 ± 24.37 µV). Post hoc analysis revealed that all the eyes in the diabetic group, including those with no DR, had a significantly reduced PhNR amplitude (P < 0.05) when compared with controls. PhNR was found to reduce in amplitude with increasing severity of DR (P < 0.05), with more significance in B/Y. Receiver operating characteristic showed highest area under the curve in B/Y PhNR (94%, P < 0.001), with maximum sensitivity and specificity of 88% and 87%, respectively. CONCLUSION: Changes in the amplitude and implicit time of ERG can reflect the severity of DR. PhNR amplitudes, especially B/Y PhNR, appear to be significantly reduced even in eyes with no DR.

Status of eye health among tribal school children in South India.

Purpose: Global trends show a high prevalence of refractive errors among children. The prevalence of vision impairment due to uncorrected refractive errors among school children is increasing and the need for management of other ocular conditions is also reported. This study presents the status of eye health and pattern of daily activities among the school children of a tribal location in Tamil Nadu, South India. Methods: A cross-sectional study was conducted in 13 schools of Karumandurai cluster, Salem district in Tamil Nadu, India. A three-phased comprehensive school screening protocol was conducted to understand the prevalence of vision impairment, refractive error, and other ocular conditions along with a survey about the daily activities of the children at school and home. Results: Among the 3655 children screened, the prevalence of vision impairment was found to be 0.62% (n = 23, 95% confidence interval [CI] 0.42-0.94) and prevalence of refractive error was 0.30% (n = 11, 95%CI 0.17-0.54), among which 0.11% (n = 4) were already wearing spectacles. A total of 44 children (1.20%; 95%CI 0.90-1.61) were found to have other ocular problems and among them, 14 (0.38%) had visual acuity less than 20/30 (6/9). Almost 84% of children required surgical or specialty eye care services. Vision impairment was more in children with other ocular conditions compared to refractive errors (P < 0.001). Conclusion: The prevalence of vision impairment and refractive errors in this tribal area was less. Ocular conditions were more prevalent than refractive errors in this tribal region with the majority of children needing specialty or surgical eye care services. This implies the need for access to secondary or tertiary eye care centers.