Biology subjects

Singh, M. S.

Publications and source records attributed to Singh, M. S..

6 recordsLinked to original sources

bioRxiv2025

A developmental transition from early neural hyperexcitability in zebrafish

Early brain development is characterized by plastic phases that play a crucial role in the establishment of functional connectivity. However, this plasticity also renders the developing brain highly sensitive to perturbations. The dynamics of the transition of the immature brain from a hyperexcitable state to a relatively stable network remains poorly understood. Using a combination of brain-wide activity mapping, behavioural and molecular analysis in response to pentylenetetrazole (PTZ), a GABAA receptor antagonist, we assessed neural hyperexcitability at different developmental stages in larval zebrafish and observed a distinct age-dependent trajectory. Specifically, we observed that early stages (4-6 dpf) exhibited high susceptibility to external perturbations, whereas by 9 dpf, both neural activity and behavioral responses were markedly attenuated. Further, upon chronic insult, rather than a cumulative increase in excitability, early life hyperexcitation events rendered the larvae resistant to subsequent induction. Put together, our work maps the temporal transition of the vertebrate brain from a state of hyperexcitability to a phase of relative resistance and has potential implications on understanding the progression of neurological conditions in the pediatric population.

neuroscience↗

bioRxiv2025

Application of machine learning to discriminate photoreceptor cell species in xenotransplanted chimeric retinas

Photoreceptor transplantation is being studied to improve visual function in retinal diseases causing blindness, such as age-related macular degeneration, hereditary eye diseases, and traumatic retinopathy, among others. Preclinical studies often involve the delivery of exogenous human photoreceptor cells into the retinas of animal models. In such experiments, a key readout is the differential frequency of donor cell somatic integration versus artificial labeling secondary to material transfer of cytosolic or nuclear labels from donor to recipient cells. For this analysis, the ability to recognize photoreceptor nuclei as being of donor (human) versus animal is key, but purely immunohistology discrimination can be challenging because of antigenic species overlap or intercellular antigen transfer. To address this challenge, we sought to develop and validate a computational technique to discriminate between photoreceptor cells of different animal species based on machine learning of nuclear morphology. Here, we aimed to evaluate the feasibility of using computer-assisted detection of separate nuclei and employing random forest classification to automate the species differentiation, among DAPI-stained photoreceptors after xeno-transplantation of human photoreceptors into the retinas of mice and pigs. Our models were trained on single-species samples and validated with mixed-species samples. We then transplanted human embryonic stem cell-derived retinal organoid cells into rodent and pig retinal degeneration models. The random forest model accurately determined cell identity post-xenotransplantation, validated by histological assessment using an anti-human nuclear antibody. Our results support the potential efficacy of employing machine learning image analysis and classification techniques that may promote experimental rigor, minimize observer bias, and enable high throughput semi-automated workflows for transplantation outcomes analysis. The methodological framework reported here may enable a more nuanced and precise analysis of the behavior of transplanted photoreceptors for the purposes of human retinal regeneration.

neuroscience↗

bioRxiv2025

Development of an image-guided non-vitrectomy subretinal access approach for trans-scleral cell and gene therapy delivery

PurposeRegenerative therapies for retinal diseases include cell and gene therapy modalities that are targeted to the subretinal space. Several recent clinical trials have shown that the morbidity of surgical access is the major limitation of safe subretinal space delivery. We aimed to develop an image-guided procedure for minimally invasive subretinal access (MISA) as a platform to deliver therapeutic agents for the treatment of degenerative retinal diseases. MethodsWe engineered prototypes of a novel common-path swept source optical coherence tomography (CP-SSOCT)-enabled needle, coaxial guide (COG), and subretinal access cannula (SAC). We pilot tested the MISA procedure in ex vivo bovine eyes and in vivo porcine ocular surgery. ResultsA- and M-mode scan recordings of ex vivo and in vivo animal eye models demonstrated that CP-SSOCT imaging from the scleral side (ab externo) was capable of identifying the retinal laminae and the sub-retinal space. We show results from in vivo porcine MISA surgeries (N=4) using the novel CP-SSOCT-enabled sub-retinal injection needle, COG, and SAC through the transscleral approach. The MISA approach enabled subretinal device placement in the posterior pole, however, cases of retinal incarceration and retinal perforation were encountered. ConclusionsWe describe a novel CP-SSOCT-guided subretinal access approach that, with further optimization, may be useful in regenerative retinal surgery.

bioengineering↗

bioRxiv2022

Cellular and Fibrillar Collagen Analyses in an Animal Model of Retinal Detachment-Related Proliferative Vitreoretinopathy Reveals a Defined Transition to Chronic Fibrosis

PurposeProliferative vitreoretinopathy (PVR) is the most common cause of failure of surgically repaired rhegmatogenous retinal detachment (RRD). Chemically-induced and cell-injection PVR models do not fully simulate the clinical characteristics of PVR in the post-RRD context. There is an unmet need for translational models in which to study mechanisms and treatments specific to RRD-PVR. MethodsRRD-PVR was induced in adult Dutch Belted rabbits. Posterior segments of enucleated globes were fixed or processed for RNA-Seq at 6 hours and 2, 7, 14, and 35 days post-induction. Histochemical staining and immunolabeling for glial fibrillary acidic protein (GFAP), alpha smooth muscle actin (SMA), vascular endothelial growth factor receptor 2 (VEGFR2), CD68, and retinal pigment epithelium 65 kDa protein (RPE65) were performed, and labeling intensity was scored. Single cell RNA sequencing was performed. ResultsAcute histopathologic changes included intravitreal and intraretinal hemorrhage, leukocytic vitritis, chorioretinitis, and retinal rarefaction. Chronic lesions showed retinal atrophy, gliosis, fibrotic subretinal membranes, and epiretinal fibrovascular proliferation. Fibrillar collagen was present in the fibrocellular and fibrovascular membranes in chronic lesions. Moderate to strong labeling of glia and vasculature was detected in chronic lesions. At day 14, most cells profiled by single cell sequencing were identified as Muller glia and microglia, consistent with immunolabeling. Expression of several fibrillar collagen genes were upregulated in chronic lesions. ConclusionsHistologic and transcriptional features of this rabbit model simulate important features of human RRD-PVR, including the transition to chronic intra and periretinal fibrosis. This high-fidelity in vivo model of RRD-PVR will enable further research on targeted treatment interventions.

pathology↗

bioRxiv2022

Transretinal migration of astrocytes and brain/spinal cord-like cells arising from transplanted human retinal organoids.

Human retinal organoid transplantation can potentially restore vision in patients with degenerative retinal diseases. How the recipient retina regulates the maturation, fate specification, and migration of transplanted organoid cells is unknown. We transplanted human retinal organoid-derived cells into photoreceptor-deficient mice, conducted histology and single-cell RNA sequencing analyses, and observed two main classes of graft-derived cells. The first class consisted of retinal astrocytes and brain/spinal cord-like neural precursors, absent or rare in cultured organoids, that migrated into all recipient retinal layers and traveled long distances. The second class consisted of retinal progenitor-derived cells, including rods and cones, that remained in the subretinal space and matured more rapidly than photoreceptors in culture. These data suggest that the recipient subretinal space promotes the maturation of transplanted photoreceptors while inducing or expanding migratory cell populations that are not normally derived from retinal progenitors. These findings have important implications for cell-based treatment of retinal diseases.

neuroscience↗

bioRxiv2021

Impaired T-cell and antibody immunity after COVID-19 infection in chronically immunosuppressed transplant recipients

Assessment of T-cell immunity to the COVID-19 coronavirus requires reliable assays and is of great interest, given the uncertain longevity of the antibody response. Some recent reports have used immunodominant spike (S) antigenic peptides and anti-CD28 co-stimulation in varying combinations to assess T-cell immunity to SARS-CoV-2. These assays may cause T-cell hyperstimulation and could overestimate antiviral immunity in chronically immunosuppressed transplant recipients, who are predisposed to infections and vaccination failures. Here, we evaluate CD154-expressing T-cells induced by unselected S antigenic peptides in 204 subjects-103 COVID-19 patients and 101 healthy unexposed subjects. Subjects included 72 transplanted and 130 non-transplanted subjects. S-reactive CD154+T-cells co-express and can thus substitute for IFN{gamma} (n=3). Assay reproducibility in a variety of conditions was acceptable with coefficient of variation of 2-10.6%. S-reactive CD154+T-cell frequencies were a) higher in 42 healthy unexposed transplant recipients who were sampled pre-pandemic, compared with 59 healthy non-transplanted subjects (p=0.02), b) lower in Tr COVID-19 patients compared with healthy transplant patients (p<0.0001), c) lower in Tr patients with severe COVID-19 (p<0.0001), or COVID-19 requiring hospitalization (p<0.05), compared with healthy Tr recipients. S-reactive T-cells were not significantly different between the various COVID-19 disease categories in NT recipients. Among transplant recipients with COVID-19, cytomegalovirus co-infection occurred in 34%; further, CMV-specific T-cells (p<0.001) and incidence of anti-receptor-binding-domain IgG (p=0.011) were lower compared with non-transplanted COVID-19 patients. Healthy unexposed transplant recipients exhibit pre-existing T-cell immunity to SARS-CoV-2. COVID-19 infection leads to impaired T-cell and antibody responses to SARS-CoV-2 and increased risk of CMV co-infection in transplant recipients.

immunology↗