Extensive Cell Loss Research Articles

Advancements and mechanisms of stem cell-based therapies for spinal cord injury in animals.

Spinal cord injury (SCI) is a neurodegenerative disorder of the central nervous system (CNS) that can lead to permanent loss of sensation and voluntary movement beyond the affected area. Extensive preclinical and clinical trials have been conducted to evaluate the safety and effectiveness of stem cells for the treatment of various CNS diseases or disorders, including SCI. However, several challenges hinder nerve cell regeneration in the injured spinal cord, such as extensive cell loss, limited neural cell regeneration capacity, axonal disruption, and the presence of growth-inhibiting molecules, particularly astroglial scarring or glial scars at the injury site in chronic cases. These obstacles pose significant challenges for physicians in restoring normal motor and sensory nerve function in both humans and animals following SCI. This review focuses on SCI pathogenesis, the mechanisms underlying the therapeutic potential of Mesenchymal Stem Cells (MSCs) in SCI, and the potential of stem cell-based therapies as promising avenues for treatment. This review article also included relevant preclinical and clinical data from animal studies.

Viral ocular surface inflammation

Viral infections of the ocular surface (OS) may cause chronic, and/or recurrent inflammation that disrupt OS homeostasis, impact quality of life, and potentially impair vision through loss of corneal transparency and/or regularity. In this presentation, we'll review basic and clinical findings underlying OS inflammation in the context of the 3 most common viral pathogens affecting the OS, i.e. Adenovirus, Herpes Simplex virus type 1 and Varicella Zoster Virus.During its acute replicative phase, adenoviral keratoconjunctivitis may trigger severe acute conjunctival inflammation, leading to transmucosal exudation and fibrinous pseudo‐membrane formation. If left untreated, the latter induce conjunctival fibrosis with extensive loss of goblet cells. In a second phase of the disease, antigen‐driven immune‐mediated mechanisms may cause chronic corneal subepithelial infiltration.In the context of HSV and VZV keratitis, OS inflammation is caused by a combination of specific immune response to viral replication, causing stromal infiltration, and neovascularisation and ii) non‐specific inflammatory processes caused by corneal innervation alterations that impact tear secretion and osmolarity. While clinical disease is most often unilateral, unaffected side may display subclinical alterations, including decrease in corneal sensitivity and corneal nerve density, and decrease in endothelial cell density.Management of viral ocular surface inflammation involves a combination of strategies aiming at controlling viral replication, decrease associated inflammatory/immune response and restore OS homeostasia.

Neutrophil extracellular traps trigger alveolar epithelial cell necroptosis through the cGAS-STING pathway during acute lung injury in mice.

Extensive loss of alveolar epithelial cells (AECs) undergoing necroptosis is a crucial mechanism of acute lung injury (ALI), but its triggering mechanism needs to be thoroughly investigated. Neutrophil extracellular traps (NETs) play a significant role in ALI. However, the effect of NETs on AECs' death has not been clarified. Our study found that intratracheal instillation of NETs disrupted lung tissue structure, suggesting that NETs could induce ALI in mice. Moreover, we observed that NETs could trigger necroptosis of AECs in vivo and in vitro. The phosphorylation levels of RIPK3 and MLKL were increased in MLE12 cells after NETs treatment (P < 0.05). Mechanistically, NETs taken up by AECs through endocytosis activated the cGAS-STING pathway and triggered AECs necroptosis. The expression of cGAS, STING, TBK1 and IRF3 were increased in MLE12 cells treated with NETs (P < 0.05). Furthermore, the cGAS inhibitor RU.521 inhibited NETs-triggered AECs necroptosis and alleviated the pulmonary damage induced by NETs in mice. In conclusion, our study demonstrates that NETs taken up by AECs via endocytosis can activate the cGAS-STING pathway and trigger AECs necroptosis to promote ALI in mice. Our findings indicate that targeting the NETs/cGAS-STING/necroptosis pathway in AECs is an effective strategy for treating ALI.

Platinum-Chimeric Carrier Cells for Ultratrace Cell Analysis in Mass Cytometry.

Mass cytometry by time-of-flight (CyTOF), a high-dimensional single-cell analysis platform, detects up to 50 biomarkers at single-cell resolution. However, CyTOF analysis of biological samples with a minimal number of available cells or rare cell subsets remains a major technical challenge due to the extensive loss of cells during cell recovery, staining, and acquisition. Here, we introduce a platinum-chimeric carrier cell strategy for mass cytometry profiling of ultratrace cell samples. Cisplatin can rapidly enter broken plasma membranes of dead cells and form a chimeric interaction with cellular proteins, peptides, and amino acids. Thus, 198Pt-cisplatin is adopted to tag carrier cells in the pretreatment stage. We investigated 8 cell lines that are commonly accessible in laboratories for their potential as carrier cells to preserve rare target cells for CyTOF analysis. We designed a panel of 35 protein biomarkers to evaluate the comprehensive single-cell subtype classification capability with or without the carrier cell strategy. We further demonstrated the detection and analysis of as few as 1 × 104 immune cells using our method. The proposed method thus allows CyTOF analysis on precious clinical samples with less abundant cells.

Reduced Cholecystokinin-Expressing Interneuron Input Contributes to Disinhibition of the Hippocampal CA2 Region in a Mouse Model of Temporal Lobe Epilepsy.

A significant proportion of temporal lobe epilepsy (TLE) patients experience drug-resistant seizures associated with mesial temporal sclerosis, in which there is extensive cell loss in the hippocampal CA1 and CA3 subfields, with a relative sparing of dentate gyrus granule cells and CA2 pyramidal neurons (PNs). A role for CA2 in seizure generation was suggested based on findings of a reduction in CA2 synaptic inhibition (Williamson and Spencer, 1994) and the presence of interictal-like spike activity in CA2 in resected hippocampal tissue from TLE patients (Wittner et al., 2009). We recently found that in the pilocarpine-induced status epilepticus (PILO-SE) mouse model of TLE there was an increase in CA2 intrinsic excitability associated with a loss of CA2 synaptic inhibition. Furthermore, chemogenetic silencing of CA2 significantly reduced seizure frequency, consistent with a role of CA2 in promoting seizure generation and/or propagation (Whitebirch et al., 2022). In the present study, we explored the cellular basis of this inhibitory deficit using immunohistochemical and electrophysiological approaches in PILO-SE male and female mice. We report a widespread decrease in the density of pro-cholecystokinin-immunopositive (CCK+) interneurons and a functional impairment of CCK+ interneuron-mediated inhibition of CA2 PNs. We also found a disruption in the perisomatic perineuronal net in the CA2 stratum pyramidale. Such pathologic alterations may contribute to an enhanced excitation of CA2 PNs and CA2-dependent seizure activity in the PILO-SE mouse model.SIGNIFICANCE STATEMENT Impaired synaptic inhibition in hippocampal circuits has been identified as a key feature that contributes to the emergence and propagation of seizure activity in human patients and animal models of temporal lobe epilepsy (TLE). Among the hippocampal subfields, the CA2 region is particularly resilient to seizure-associated neurodegeneration and has been suggested to play a key role in seizure activity in TLE. Here we report that perisomatic inhibition of CA2 pyramidal neurons mediated by cholecystokinin-expressing interneurons is selectively reduced in acute hippocampal slices from epileptic mice. Parvalbumin-expressing interneurons, in contrast, appear relatively conserved in epileptic mice. These findings advance our understanding of the cellular mechanisms underlying inhibitory disruption in hippocampal circuits in a mouse model of spontaneous recurring seizures.

Abstract PR009: Basal cell lineage plasticity in prostate homeostasis, repair and tumor initiation

Abstract Androgen deprivation is the paradigm of prostate cancer treatment. It is often linked to the loss of luminal cells, but the basal cell fates under androgen deprivation, prostatitis and cancer initiation remain unclear. Using a genetic proliferation tracer (ProTracer) system, we found that adult prostate basal cells also had extensive cell loss and proliferation during androgen-mediated prostate regression and regeneration. A rare basal population that expresses luminal cell genes (termed Basal-B) and a large basal population (termed Basal-A) were identified in the mouse prostate by single-cell RNA sequencing. Basal-B cells exhibited greater capacity for organoid formation and luminal cell differentiation in vitro. Basal-A and Basal-B cells were specifically tracked at the same time in vivo using a genetic lineage-tracing system that uses dual recombinases (Cre and Dre). Fate-mapping showed that prostate basal regeneration is not driven by rare Basal-B cells. Clonal analysis showed that Basal-B cells had the better tendency to generate luminal cells under bacteria-induced prostatitis. Moreover, oncogenic transformation of basal cells promoted the Basal-B cell marker gene expression in vivo. This study provides genetic evidence of a bona fide of basal cell lineage plasticity under androgen deprivation, prostatitis and oncogenic transformation contexts. Citation Format: Dong Gao. Basal cell lineage plasticity in prostate homeostasis, repair and tumor initiation [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr PR009.

Supporting-cell vs. hair-cell survival in the human cochlea: Implications for regenerative therapies

Animal studies have shown that the supporting-cells surviving in the organ of Corti after cochlear insult can be transdifferentiated into hair cells as a treatment for sensorineural hearing loss. Clinical trials of small-molecule therapeutics have been undertaken, but little is known about how to predict the pattern and degree of supporting-cell survival based on audiogram, hearing loss etiology or any other metric obtainable pre-mortem. To address this, we systematically assessed supporting-cell and hair cell survival, as a function of cochlear location in 274 temporal bone cases from the archives at the Massachusetts Eye and Ear and compared the histopathology with the audiograms and hearing-loss etiologies. Results showed that supporting-cell survival was always significantly greater in the apical half than the basal half of the cochlea, that inner pillars were more robust than outer pillars or Deiters’ cells, and that total replacement of all supporting cells with a flat epithelium was rare outside of the extreme basal 20% of the cochlea. Supporting cell survival in the basal half of the cochlea was better correlated with the slope of the audiogram than with the mean high-frequency threshold per se: i.e. survival was better with flatter audiograms than with steeply down-sloping audiograms. Cochlear regions with extensive hair cell loss and exceptional supporting cell survival were most common in cases with hearing loss due to ototoxic drugs. Such cases also tended to have less pathology in other functionally critical structures, i.e. spiral ganglion neurons and the stria vascularis.

Retinitis pigmentosa-associated mutations in mouse Prpf8 cause misexpression of circRNAs and degeneration of cerebellar granule cells.

A subset of patients with retinitis pigmentosa (RP) carry mutations in several spliceosomal components including the PRPF8 protein. Here, we established two alleles of murine Prpf8 that genocopy or mimic aberrant PRPF8 found in RP patients-the substitution p.Tyr2334Asn and an extended protein variant p.Glu2331ValfsX15. Homozygous mice expressing the aberrant Prpf8 variants developed within the first 2 mo progressive atrophy of the cerebellum because of extensive granule cell loss, whereas other cerebellar cells remained unaffected. We further show that a subset of circRNAs were deregulated in the cerebellum of both Prpf8-RP mouse strains. To identify potential risk factors that sensitize the cerebellum for Prpf8 mutations, we monitored the expression of several splicing proteins during the first 8 wk. We observed down-regulation of all selected splicing proteins in the WT cerebellum, which coincided with neurodegeneration onset. The decrease in splicing protein expression was further pronounced in mouse strains expressing mutated Prpf8. Collectively, we propose a model where physiological reduction in spliceosomal components during postnatal tissue maturation sensitizes cells to the expression of aberrant Prpf8 and the subsequent deregulation of circRNAs triggers neuronal death.

Blood biomarkers in ALS: challenges, applications and novel frontiers.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease among adults. With diagnosis reached relatively late into the disease process, extensive motor cell loss narrows the window for therapeutic opportunities. Clinical heterogeneity in ALS and the lack of disease-specific biomarkers have so far led to large-sized clinical trials with long follow-up needed to define clinical outcomes. In advanced ALS patients, there is presently limited scope to use imaging or invasive cerebrospinal fluid (CSF) collection as a source of disease biomarkers. The development of more patient-friendly and accessible blood biomarker assays is hampered by analytical hurdles like the matrix effect of blood components. However, blood also provides the opportunity to identify disease-specific adaptive changes of the stoichiometry and conformation of target proteins and the endogenous immunological response to low-abundance brain peptides, such as neurofilaments (Nf). Among those biomarkers under investigation in ALS, the change in concentration before or after diagnosis of Nf has been shown to aid prognostication and to allow the a priori stratification of ALS patients into smaller sized and clinically more homogeneous cohorts, supporting more affordable clinical trials. Here, we discuss the technical hurdles affecting reproducible and sensitive biomarker measurement in blood. We also summarize the state of the art of non-CSF biomarkers in the study of prognosis, disease progression, and treatment response. We will then address the potential as disease-specific biomarkers of the newly discovered cryptic peptides which are formed down-stream of TDP-43 loss of function, the hallmark of ALS pathobiology.

Alterations in the jejunal microbiota and fecal metabolite profiles of rabbits infected with Eimeria intestinalis

BackgroundRabbit coccidiosis is a major disease caused by various Eimeria species and causes enormous economic losses to the rabbit industry. Coccidia infection has a wide impact on the gut microbiota and intestinal biochemical equilibrium. In the present study, we established a model of Eimeria intestinalis infection in rabbits to evaluate the jejunal microbiota and fecal metabolite profiles.MethodsRabbits in the infected group were orally inoculated with 3 × 103E. intestinalis oocysts. On the eighth day of infection, jejunal contents and feces were collected for 16S rRNA gene sequencing and liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis, respectively. Jejunum tissues were harvested for hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), and immunohistochemistry (IHC) staining.ResultsHistopathological analysis showed that the whole jejunum was parasitized by E. intestinalis in a range of life cycle stages, and PAS staining showed that E. intestinalis infection caused extensive loss of goblet cells. IHC staining revealed that TNF-α expression was higher in the E. intestinalis infection group. Moreover, both the jejunal microbiota and metabolites significantly altered after E. intestinalis infection. At the genus level, the abundances of Escherichia and Enterococcus significantly increased in the infected group compared with the control group, while those of Oscillospira, Ruminococcus, Bacteroides, Akkermansia, Coprococcus, and Sarcina significantly decreased. In addition, 20 metabolites and two metabolic pathways were altered after E. intestinalis infection, and the major disrupted metabolic pathway was lipid metabolism.ConclusionsEimeria intestinalis infection induced intestinal inflammation and destroyed the intestinal homeostasis at the parasitized sites, leading to significant changes in the gut microbiota and subsequent corresponding changes in metabolites.Graphical

Targeted disruption of Gdi2 causes early embryonic lethality

IntroductionGDI2 regulates the GDP/GTP exchange reaction of Rab proteins by inhibiting the dissociation of GDP and the subsequent binding of GTP, dysregulation of GDI2 has been reported in many different cancers. Recently, we found that GDI2 bound to the ITIM domain of Siglec-G under normal homeostasis, whereas Rab1a was recruited to the ITIM domain during bacterial infection. Therefore, GDI2 and Rab1a may regulate the immune response through interaction with the ITIM domain during bacterial infection. However, the regulation of the inflammatory response by GDI2 in vivo and its regulatory mechanism remain unknown. MethodsWe generated a Gdi2 null mutant mouse with a trapped Gdi2 gene and examined the expression by X-gal and immunohistochemistry staining. TUNEL staining was used to determine the apoptosis cells. ResultsHere we show that Gdi2 is essential for embryonic development. One functional Gdi2 allele is sufficient for murine embryo development, but complete loss of Gdi2 leads to embryonic lethality. Developmental retardation of Gdi2−/− mice is apparent at E10.5 to E14.5, with no viable Gdi2−/− embryos detected after E14.5. Histological analysis revealed extensive cell death and cell loss in Gdi2−/− embryos. Apoptosis was confirmed by staining with cleaved caspase-3, suggesting that Gdi2 maintain homeostasis by regulating the apoptosis of the cells. There was no significant difference in cytokine production and survival between wild-type and Gdi2+/− mice after LPS challenge. DiscussionThese findings suggest that one Gdi2 allele is sufficient to maintain function. However, the detailed molecular mechanism underlying Gdi2 in regulating the embryonic development needs further identification.

Deafness in an auditory specialist, the big brown bat (Eptesicus fuscus)

Bats are long-lived animals that show presumed resistance to noise-induced and age-related hearing loss, which has been attributed to their dependence on sound processing for survival. Echolocation and basic auditory functions have been studied extensively in the big brown bat (Eptesicus fuscus), an insectivorous microchiropteran species. We conducted hearing tests and analysis of cochlear sensory cells in a group of big brown bats that exhibited anomalies in behavioral sonar tracking experiments and/or lacked neural responses to acoustic stimulation in subcortical auditory nuclei. We show for the first time the presence of profound deafness and extensive cochlear damage in an echolocating bat species. Auditory brainstem responses were abnormal or absent in these bats, and histological analyses of their cochleae revealed extensive loss of hair cells, supporting cells, and spiral ganglion neurons. The underlying cause of deafness is unknown.

GJB2 gene therapy and conditional deletion reveal developmental stage-dependent effects on inner ear structure and function

Pathogenic variants in GJB2, the gene encoding connexin 26, are the most common cause of autosomal-recessive hereditary deafness. Despite this high prevalence, pathogenic mechanisms leading to GJB2-related deafness are not well understood, and cures are absent. Humans with GJB2-related deafness retain at least some auditory hair cells and neurons, and their deafness is usually stable. In contrast, mice with conditional loss of Gjb2 in supporting cells exhibit extensive loss of hair cells and neurons and rapidly progress to profound deafness, precluding the application of therapies that require intact cochlear cells. In an attempt to design a less severe Gjb2 animal model, we generated mice with inducible Sox10iCreERT2-mediated loss of Gjb2. Tamoxifen injection led to reduced connexin 26 expression and impaired function, but cochlear hair cells and neurons survived for 2 months, allowing phenotypic rescue attempts within this time. AAV-mediated gene transfer of GJB2 in mature mutant ears did not demonstrate threshold improvement and in some animals exacerbated hearing loss and resulted in hair cell loss. We conclude that Sox10iCreERT2;Gjb2flox/flox mice are valuable for studying the biology of connexin 26 in the cochlea. In particular, these mice may be useful for evaluating gene therapy vectors and development of therapies for GJB2-related deafness.

Critical role of interferons in gastrointestinal injury repair

The etiology of ulcerative colitis is poorly understood and is likely to involve perturbation of the complex interactions between the mucosal immune system and the commensal bacteria of the gut, with cytokines acting as important cross-regulators. Here we use IFN receptor-deficient mice in a dextran sulfate sodium (DSS) model of acute intestinal injury to study the contributions of type I and III interferons (IFN) to the initiation, progression and resolution of acute colitis. We find that mice lacking both types of IFN receptors exhibit enhanced barrier destruction, extensive loss of goblet cells and diminished proliferation of epithelial cells in the colon following DSS-induced damage. Impaired mucosal healing in double IFN receptor-deficient mice is driven by decreased amphiregulin expression, which IFN signaling can up-regulate in either the epithelial or hematopoietic compartment. Together, these data underscore the pleiotropic functions of IFNs and demonstrate that these critical antiviral cytokines also support epithelial regeneration following acute colonic injury.

The Effect of Autologous Adipose Tissue–Derived Mesenchymal Stem Cells’ Therapy in the Treatment of Chronic Posttraumatic Spinal Cord Injury in a Domestic Ferret Patient

Spinal cord injury (SCI) is considered as one of the most problematic neurological conditions requiring specialized clinical intervention. Taking into account that SCI is characterized by extensive loss of nerve cells, stem cell–based therapy seems to be a reasonable modern strategy to the treatment of SCI. The presented case report describes for the first time experimental treatment with the use of autologous adipose tissue–derived mesenchymal stem cells (ADSCs) of the chronic posttraumatic SCI in a domestic ferret patient with paresis of back legs. It should be noted that most reports in the available literature concern ADSC-based therapies for acute or subacute SCI treatment in other species. Application of ADSC-based therapy did not cause any adverse reactions and resulted in significant improvement of neurological and motor functions. Based on these outcomes, it may be concluded that this form of therapy is promising and may be potentially translated into clinical veterinary practice.

Dazl determines primordial follicle formation through the translational regulation of Tex14.

Deleted in azoospermia-like (DAZL) is a germ cell RNA-binding protein that is essential for entry and progression through meiosis. The phenotype of the Dazl knockout mouse has extensive germ cell loss because of incomplete meiosis. We have created a Dazl hypomorph model using short interfering RNA knockdown in mouse fetal ovary cultures, allowing investigation of Dazl function in germ cell maturation. Dazl hypomorph ovaries had a phenotype of impaired germ cell nest breakdown with a 66% reduction in total follicle number and an increase in the proportion of primordial follicles (PMFs), with smaller oocytes within these follicles. There was no significant early germ cell loss or meiotic delay. Immunostaining of intercellular bridge component testis-expressed protein (Tex)14 showed ∼59% reduction in foci number and size, without any change in Tex14 mRNA levels. TEX14 expression was also confirmed in the human fetal ovary across gestation. Using 3'UTR-luciferase reporter assays, translational regulation of TEX14 was demonstrated to be DAZL-dependant. Dazl is therefore essential for normal intercellular bridges within germ cell nests and their timely breakdown, with a major impact on subsequent assembly of PMFs.-Rosario, R., Crichton, J. H., Stewart, H. L., Childs, A. J., Adams, I. R., Anderson, R. A. Dazl determines primordial follicle formation through the translational regulation of Tex14.

Transgenic Archaerhodopsin-3 Expression in Hypocretin/Orexin Neurons Engenders Cellular Dysfunction and Features of Type 2 Narcolepsy.

Narcolepsy, characterized by excessive daytime sleepiness, is associated with dysfunction of the hypothalamic hypocretin/orexin (Hcrt) system, either due to extensive loss of Hcrt cells (Type 1, NT1) or hypothesized Hcrt signaling impairment (Type 2, NT2). Accordingly, efforts to recapitulate narcolepsy-like symptoms in mice have involved ablating these cells or interrupting Hcrt signaling. Here, we describe orexin/Arch mice, in which a modified archaerhodopsin-3 gene was inserted downstream of the prepro-orexin promoter, resulting in expression of the yellow light-sensitive Arch-3 proton pump specifically within Hcrt neurons. Histological examination along with ex vivo and in vivo electrophysiological recordings of male and female orexin/Arch mice demonstrated silencing of Hcrt neurons when these cells were photoilluminated. However, high expression of the Arch transgene affected cellular and physiological parameters independent of photoillumination. The excitability of Hcrt neurons was reduced, and both circadian and metabolic parameters were perturbed in a subset of orexin/Arch mice that exhibited high levels of Arch expression. Orexin/Arch mice also had increased REM sleep under baseline conditions but did not exhibit cataplexy, a sudden loss of muscle tone during wakefulness characteristic of NT1. These aberrations resembled some aspects of mouse models with Hcrt neuron ablation, yet the number of Hcrt neurons in orexin/Arch mice was not reduced. Thus, orexin/Arch mice may be useful to investigate Hcrt system dysfunction when these neurons are intact, as is thought to occur in narcolepsy without cataplexy (NT2). These results also demonstrate the utility of extended phenotypic screening of transgenic models when specific neural circuits have been manipulated.SIGNIFICANCE STATEMENT Optogenetics has become an invaluable tool for functional dissection of neural circuitry. While opsin expression is often achieved by viral injection, stably integrated transgenes offer some practical advantages. Here, we demonstrate successful transgenic expression of an inhibitory opsin in hypocretin/orexin neurons, which are thought to promote or maintain wakefulness. Both brief and prolonged illumination resulted in inhibition of these neurons and induced sleep. However, even in the absence of illumination, these cells exhibited altered electrical characteristics, particularly when transgene expression was high. These aberrant properties affected metabolism and sleep, resulting in a phenotype reminiscent of the narcolepsy Type 2, a sleep disorder for which no good animal model currently exists.

Musashi-2 and related stem cell proteins in the mouse suprachiasmatic nucleus and their potential role in circadian rhythms

BackgroundThe suprachiasmatic nucleus (SCN) of the mammalian hypothalamus contains the master circadian clock of the body and an unusually large number of cells expressing stem cell-related proteins. These seemingly undifferentiated cells may serve in entrainment of the SCN circadian clock to light cycles or allow it to undergo neural plasticity important for modifying its rhythmic output signals. These cells may also proliferate and differentiate into neurons or glia in response to episodic stimuli or developmental events requiring alterations in the SCN’s control of physiology and behavior. ProblemTo characterize expression of stem cell related proteins in the SCN and the effects of stem-like cells on circadian rhythms. MethodsExplant cultures of mouse SCN were maintained in medium designed to promote survival and growth of stem cells but not neuronal cells. Several stem cell marker proteins including SRY-box containing gene 2 (SOX2), nestin, vimentin, octamer-binding protein 4 (OCT4), and Musashi RNA-binding protein 2 (MSI2) were identified by immunocytochemistry in histological sections from adult mouse SCN and in cultures of microdissected SCN. A bioinformatics analysis located potential SCN targets of MSI2 and related RNA-binding proteins. ResultsCells expressing stem cell markers proliferated in culture. Immunostained brain sections and bioinformatics supported the view that MSI2 regulates immature properties of SCN neurons, potentially providing flexibility in SCN neural circuits. Explant cultures had ongoing mitotic activity, indicated by proliferating-cell nuclear antigen, and extensive cell loss shown by propidium iodide staining. Cells positive for vasoactive intestinal polypeptide (VIP) that are highly enriched in the SCN were diminished in explant cultures. Despite neuronal cell loss, tissue remained viable for over 7 weeks in culture, as shown by bioluminescence imaging of explants prepared from SCN of Per1::luc transgenic mice. The circadian rhythm in SCN gene expression persisted in brain slice cultures in stem cell medium. Prominent, widespread expression of RNA-binding protein MSI2 supported the importance of posttranscriptional regulation in SCN functions and provided further evidence of stem-like cells. ConclusionThe results show that the SCN retains properties of immature neurons and these properties persist in culture conditions suitable for stem cells, where the SCN stem-like cells also proliferate. These properties may allow adaptive circadian rhythm adjustments. Further exploration should examine stem-like cells of the SCN in vivo, how they may affect circadian rhythms, and whether MSI2 serves as a master regulator of SCN stem-like properties.

Cajal Cell Counts are Important Predictors of Outcomes in Drug Refractory Gastroparesis Patients With Neurostimulation.

Cajal cells serve as the pacemaker cells of the gastrointestinal tract and regulates peristalsis. On the baisis of that fact, it has been hypothesized that a decrease in Cajal cells can lead to gastroparesis and other motility issues. Treatment with medications has a limited efficacy and most resort to gastric electrical stimulation (GES) devices for symptomatic relief. We believe that the number of Cajal cells present is directly proportional to symptomatic relief with GES. Twenty-three (white female) subjects were recruited from the gastric motility clinic University of Mississipi for this study with the criteria of drug refractory gastropersis. Symptoms were measured using Likert scale and gastric emptying times were measured pre-GES and post-GES. Serosal electrogram measurements were recorded during surgical placement of permanent electrical stimulator under various modes. Cajal cell count scoring via immunohistochemistry were performed during the implantaion of the GES. The data were grouped in 2 categories based on the Cajal cells that is ≥2.00 and <2.00. Subjects with higher Cajal cells reported a statiscially improvement in gastroperesis symptoms. Significant differences were also noted in the first hour gastric emptying study. The mean group difference is 17.5 (95% confidence interval, 1.41-33.58; P=0.035). Serosal amplitude differences were noted being significantly higher in the group with ≥2 cajal cells. Electrograms obtained after GES demonstrates immediate improvement in gastric electrical activity and gastroparesis symptoms in patients with relatively higher Cajal cell counts when compared with patients with extensive loss of Cajal cells.

Heat Induced Oxidative Stress and Aberrations in Liver Function Leading to Hepatic Injury in Rats

Exposure to heat stress (HS) elicits systemic and cellular response in experimental animals and humans. The current study was undertaken to determine the effect of HS on liver microstructure and function in rats. A heat simulation chamber with ambient temperature (Ta) 45 ± 0.5 °C and relative humidity (RH) 30 ± 5 per cent was used to expose animals to HS. Rats were categorised as moderately heat stressed (MHS, Tc = 40 °C) and severely heat stressed (SHS, Tc = 42 °C) group. We observed that with rise in core temperature (Tc) alanine aminotransferase(ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) levels were increased but glucose level was decreased in both plasma and liver tissue. Significantly elevated levels of reactive oxygen species (ROS) and nitric oxide (NO) were detected in liver of MHS and SHS animals. Additionally, glutathione disulfide and glutathione (GSSG and GSH) ratio was found to be increased with rise in Tc which suggested saturation in antioxidant capacity of tissue. Furthermore, levels of heat shock proteins (HSPs) and caspases were upregulated upon HS. Results of histological examination indicated extensive loss of cells in liver parenchyma leading to disorganisation of lobular structure. Thus, biochemical and histological studies in experimental animals demonstrates that HS may severely altered structural and biochemical functions of liver.