Cortical Explants Research Articles

Ultrasound-Mediated Lysozyme Microbubbles Targeting NOX4 Knockdown Alleviate Cisplatin-Exposed Cochlear Hair Cell Ototoxicity.

The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) protein plays an essential role in the cisplatin (CDDP)-induced generation of reactive oxygen species (ROS). In this study, we evaluated the suitability of ultrasound-mediated lysozyme microbubble (USMB) cavitation to enhance NOX4 siRNA transfection in vitro and ex vivo. Lysozyme-shelled microbubbles (LyzMBs) were constructed and designed for siNOX4 loading as siNOX4/LyzMBs. We investigated different siNOX4-based cell transfection approaches, including naked siNOX4, LyzMB-mixed siNOX4, and siNOX4-loaded LyzMBs, and compared their silencing effects in CDDP-treated HEI-OC1 cells and mouse organ of Corti explants. Transfection efficiencies were evaluated by quantifying the cellular uptake of cyanine 3 (Cy3) fluorescein-labeled siRNA. In vitro experiments showed that the high transfection efficacy (48.18%) of siNOX4 to HEI-OC1 cells mediated by US and siNOX4-loaded LyzMBs significantly inhibited CDDP-induced ROS generation to almost the basal level. The ex vivo CDDP-treated organ of Corti explants of mice showed an even more robust silencing effect of the NOX4 gene in the siNOX4/LyzMB groups treated with US sonication than without US sonication, with a marked abolition of CDDP-induced ROS generation and cytotoxicity. Loading of siNOX4 on LyzMBs can stabilize siNOX4 and prevent its degradation, thereby enhancing the transfection and silencing effects when combined with US sonication. This USMB-derived therapy modality for alleviating CDDP-induced ototoxicity may be suitable for future clinical applications.

Induction of cell death by sodium hexachloroplatinate (IV) in the HEI-OC1 cell line, primary rat spiral ganglion cells and rat organ of Corti explants.

Although cochlear implants have become a well-established method for patients with sensory neural hearing loss, clinical results indicate that in some cases, corrosion of electrode contacts leads to high impedance that interferes with successful stimulation of the auditory nerve. As it is unclear whether corrosion products induce cell damage, we focused on cell culture models of the organ of Corti cell line (HEI-OC1), rat spiral ganglion cells (SGC) and rat organ of Corti explant (OCex) cultivated from neonatal rat cochleae to characterize the cytotoxicity of sodium hexachloroplatinate (IV) (Na2(PtCl6)). The oxidative activity in HEI-OC1 cells decreased with increasing Na2(PtCl6) concentrations between 8 and 16 ng/μl, and live cell staining with Calcein acetoxymethyl/Ethidium homodimer III revealed an increasing number of cells with disrupted membranes. Ultrastructural evidence of mitophagy followed by necroptosis was detected. Additionally, exposure of the SGC to 15-35 ng/μl Na2(PtCl6) dose-dependently reduced neuronal survival and neuritogenesis, as determined by neurofilament antigen staining. In parallel, staining glial cells and fibroblasts with specific antibodies confirmed the dose-dependent induction of cell death by Na2(PtCl6). Exposure of the OCex to 25-45 ng/μl Na2(PtCl6) resulted in severe concentration-dependent hair cell loss. Our data show for the first time that Na2(PtCl6) induces cell death in a concentration-dependent manner in inner ear cell types and tissues.

Berberine protects against neomycin-induced ototoxicity by reducing ROS generation and activating the PI3K/AKT pathway

In mammals, aminoglycoside antibiotic-induced injury to hair cells (HCs) and associated spiral ganglion neurons (SGNs) is irreversible and eventually leads to permanent hearing loss. Efforts have been directed towards the advancement of efficacious therapeutic treatments to protect hearing loss, but the ideal substance for treating the damaged cochlear sensory epithelium has yet to be identified. Berberine (BBR), a quaternary ammonium hydroxide extracted from Coptis chinensis, has been found to display potential anti-oxidant and neuroprotective properties. However, its involvement in aminoglycoside antibiotic-induced ototoxicity has yet to be explored or assessed. In the present study, we explored the possible anti-oxidative properties of BBR in mitigating neomycin-triggered ototoxicity. An improved survival of HCs and SGN nerve fibers (NFs) in organ of Corti (OC) explants after neomycin with BBR co-treatment was observed, and BBR treatment attenuated reactive oxygen species (ROS) generation and reduced cleaved caspase-3 signaling by activating six phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling relative subtypes, and the addition of PI3K/AKT suppressor LY294002 resulted in a decrease in the protective effect. The protective effect of BBR against ototoxicity was also evident in a neomycin-injured animal model, as evidenced by the preservation of HC and SGN in mice administered subcutaneous BBR for 7 days. In summary, all results suggest that BBR has potential as a new and effective otoprotective agent, operating via the PI3K/AKT signaling pathway.

Exogenous humanin and MOTS-c function as protective agents against gentamicin-induced hair cell damage

Loss of hair cells can lead to irreversible sensorineural hearing loss. Therefore, hair cell preservation is critical for hearing. Mitochondrial derived peptides (MDPs) are bioactive peptides and prominent members of this family are humanin (HN) and the mitochondrial-open-reading frame of the twelve S c (MOTS-c). The protective roles of HN and MOTS-c in age-related diseases and in various tissues exposed to cellular stresses have been demonstrated. The involvement of MDPs in the inner ear remains to be investigated. Therefore, we determined the expression of rattin, the homolog of humanin, in inner ear tissues. Then, we found that HN and MOTS-c showed a significant protective effect on hair cells in organ of Corti explants exposed to gentamicin. Treatment with HN decreased gentamicin-induced phosphorylation of AKT, whereas treatment with MOTS-c increased phosphorylation of AMPKα in explants. Our data indicate that MDPs exert a protective function in gentamicin-induced hair cell damage. Therefore, MDPs may contribute to design new preventive strategies against hearing loss.

Cochlear Explantation Prevents Hair Cell Degeneration in Transmembrane Serine Protease 3 (Tmprss3) Deficient Mice

Background/Objective: Transmembrane serine protease 3 (TMPRSS3) is the most common causative hearing loss gene in adults undergoing cochlear implantation and a significant cause of non-syndromic hearing loss. However, the function of TMPRSS3 within the inner ear is unknown. In murine models, Tmprss3Y260X/Y260X mutants (Tmprss3-/-) have normal hair cell development followed by rapid hair cell degeneration from postnatal day 12 (P12) to P14. The endocochlear potential in vivo rises from P7 to a peak at P12, when mice begin to hear, which temporally corresponds to the HC degeneration in Tmprss3-/- mice. We tested if hair cell death occurs after removal of the endocochlear potential through cochlear explantation.Methods: P7 organ of Corti explants from control (Tmprss3+/-) and Tmprss3-/- mice were cultured in 5 mM potassium solution for 7 days in vitro (equivalent to P14). Whole mounts of cultured (P7+7DIV) and in vivo P14 cochlea were immunostained for MYO7A (hair cell marker) and DAPI followed by quantification of inner and outer hair cell (IHC/OHC) counts per 20 μm. Statistical analysis included two tailed t-test with p-value of (<0.01, n=6).Results: As expected, there is complete loss of hair cells at P14 in vivo in Tmprss3-/- mice. Compared to P14 in vivo, Tmprss3-/- OC explants displayed significantly improved IHC and OHC survival (P<0.001). IHC and OCH survival was similar between control and Tmprss3-/- OHC explants in vitro (P=0.99, n=6)Conclusion: These results suggest that degeneration of Tmprss3-/- hair cells is due to factors related to the endocochlear potential and implicate TMPRSS3 function in regulation of epithelial tight junctions. Future directions include confirming that hair cell death is potassium-mediated by crossing Tmprss3-/- mice with Pou3f4del-J mice, which have a decreased endocochlear potential without hair cell degeneration.

Understanding the effects of opioids vs non-opioids in the treatment of neonatal abstinence syndrome, an in vitro model.

Neonatal abstinence syndrome (NAS) refers to cadre of withdrawal manifestations in infants born to mothers who used illicit and licit substances during pregnancy. The increasing prevalence of NAS has been largely due to the maternal use of opioids during pregnancy. NAS contributes to increased morbidity and long-term disability in surviving infants. Clinically, oral opioid therapies for opioid exposure have been a standard treatment with morphine (MO) being the most commonly used medication. Recently, a non-opioid agent, clonidine (CD) has also been used with potentially favorable short- and long-term outcomes in infants. However, data regarding the cellular and molecular effects of these treatments on the developing brain is still lacking due to a lack of a reliable animal model that targets the neonatal brain. To address this gap in knowledge we determined the effects of MO or CD on the cell death of neonatal cortical explant cultures that were exposed to oxycodone (OXY) in utero. Sprague Dawley rats were randomized and implanted with programmable infusion pumps before mating to receive either the OXY (dose increasing from 1.21-1.90 mg/kg/day to a maximum dose of 2.86-3.49 mg/kg/day) or normal saline (NS) throughout pregnancy and until one week after delivery. Male and female rat pups were sacrificed on postnatal day 4, and the prefrontal cortex (PFC) and hippocampus (HC) were dissected and treated with MO (0.10-1.00 µM) or CD (1.20-120.00 µM) in culture media. After 5 days of treatment the explants were labeled with propidium iodide to detect cell death. Dead cells were analyzed and counted under fluorescence microscopy. In explants from the PFC, cell death was greater in those prenatally exposed to OXY and postnatally treated with MO (OXY/MO) (736.8 ± 76.5) compared to OXY/CD (620.9 ± 75.0; p = 0.005). In the HC explants, mean cell death counts were not significantly different between groups regardless of prenatal exposure or postnatal treatment (p = 0.19). The PFC is vital in controlling higher-order executive functions such as behavioral flexibility, learning and working memory. Therefore, our finding is consistent with executive function problems in children with prenatal opioid exposure.

The protective effect of rutin against the cisplatin-induced cochlear damage in vitro

Rutin is a natural flavonoid, with typical effects including interaction with enzymes and scavenging of free radicals. However, the role of rutin in the auditory system is still unclear. In the present study, we used neonatal organ of Corti explants in vitro to investigate the effects of rutin in cisplatin-induced ototoxicity. The TUNEL assay and the cleaved caspase-3 immunohistochemistry were used to detect the apoptosis of hair cell (HCs) in cochlear explants, and the MitoSox-Red staining was used to detect the difference in mitochondrial superoxide among different groups. Western blot was used to investigate the expression of genes. Confocal microscopy showed that after pretreatment with rutin, the accumulation of reactive oxygen species in HCs caused by cisplatin exposure was significantly reduced. Furthermore, the expression levels of p-P38 and p-JNK were significantly decreased, while ratio of p-AKT/AKT was significantly upregulated. Our study showed that rutin reduced cisplatin-induced HCs death in neonatal cochlear explants in vitro. The potential mechanism involved the alleviation of mitochondrial damage, the scavenging of reactive oxygen species (ROS), the suppression of MAPK signaling pathway, and the activation of PI3K/AKT signaling pathway.

Regulation of membrane homeostasis by TMC1 mechanoelectrical transduction channels is essential for hearing

The mechanoelectrical transduction (MET) channel complex of auditory hair cells converts sound into electrical signals, allowing us to hear. After decades of research, the transmembrane-like channel 1 and 2 (TMC1 and TMC2) have been recently identified as pore-forming subunits of the MET channels, but the molecular peculiarity that differentiates these two proteins and makes TMC1 essential for hearing remains elusive. Inspired by the evolutionary and structural relation between the TMC proteins and the TMEM16 lipid scramblases, we investigated the potential role of TMC proteins in the regulation of hair cell membrane homeostasis using Airyscan superresolution confocal microscopy and organ of Corti explants from 13 different transgenic mouse lines.

A combinatorial approach to protect sensory tissue against cisplatin-induced ototoxicity

Sensorineural Hearing Loss (SNHL) is a highly prevalent disorder involving permanent damage or loss to the inner ear's mechano-sensory hair cells and nerve fibers. Major contributing causes are ototoxic drugs, loud noises, and aging. Drug-induced hearing loss (DIHL), affects over 25% of patients treated with common therapeutics such as aminoglycoside antibiotics, loop diuretics or chemotherapeutics. A commonly used chemotherapeutic agent, cisplatin, is very effective for treating malignant tumors, but results in a majority of patients experiencing irreversible hearing loss and/or tinnitus. Additionally, since there is currently no FDA-approved treatments for SNHL, attenuation of ototoxicity is a major area of investigation in oncology, otolaryngology and hearing research. Several potential otoprotective agents have been investigated at the clinical trial stage, but none have progressed to a full FDA-approval. In this study, we investigated a combinatorial approach comprised of an antioxidant, a p53 inhibitor and a neurotrophin, as a multifactorial otoprotective treatment for cisplatin exposure. In vitro, HEI-OC1 cells, an immortalized organ of Corti epithelial cell line, pre-treated with this biotherapeutic cocktail had significantly reduced cisplatin-induced cell death, DNA fragmentation, and apoptotic activation. In an ex vivo study, rat pup D2-D3 organ of Corti explants, significant protection against cisplatin-based hair cell and neuronal loss was achieved by delivery of the same combinatorial pretreatment. Interestingly, the hair cell protection was localized to the basal and middle regions of the organ of Corti. Together, these findings highlight a novel approach to attenuate cisplatin ototoxicity and potentially prevent DIHL by addressing biological mechanisms of cisplatin ototoxicity.

A deep learning approach to quantify auditory hair cells

Hearing loss affects millions of people worldwide. Yet, there are still no curative therapies for sensorineural hearing loss. Frequent causes of sensorineural hearing loss are due to damage or loss of the sensory hair cells, the spiral ganglion neurons, or the synapses between them. Culturing the organ of Corti allows the study of all these structures in an experimental model, which is easy to manipulate. Therefore, the in vitro culture of the neonatal mammalian organ of Corti remains a frequently used experimental system, in which hair cell survival is routinely assessed. However, the analysis of the surviving hair cells is commonly performed via manual counting, which is a time-consuming process and the inter-rater reliability can be an issue.Here, we describe a deep learning approach to quantify hair cell survival in the murine organ of Corti explants. We used StarDist, a publicly available platform and plugin for Fiji (Fiji is just ImageJ), to train and apply our own custom deep learning model. We successfully validated our model in untreated, cisplatin, and gentamicin treated organ of Corti explants. Therefore, deep learning is a valuable approach for quantifying hair cell survival in organ of Corti explants. Moreover, we also demonstrate how the publicly available Fiji plugin StarDist can be efficiently used for this purpose.

Effects of Moringa Extract on Aminoglycoside-Induced Hair Cell Death and Organ of Corti Damage.

Moringa extract, a naturally occurring anti-oxidant, protects against aminoglycoside-induced hair cell death and hearing loss within the organ of Corti. Reactive oxygen species (ROS) arise primarily in the mitochondria and have been implicated in aminoglycoside-induced ototoxicity. Mitochondrial dysfunction results in loss of membrane potential, release of caspases, and cell apoptosis. Moringa extract has not previously been examined as a protective agent for aminoglycoside-induced ototoxicity. Putative otoprotective effects of moringa extract were investigated in an organotypic model using murine organ of Corti explants subjected to gentamicin-induced ototoxicity. Assays evaluated hair cell loss, cytochrome oxidase expression, mitochondrial membrane potential integrity, and caspase activity. In vitro application of moringa conferred significant protection from gentamicin-induced hair cell loss at dosages from 25 to 300 μg/mL, with dosages above 100 μg/mL conferring near complete protection. Assays demonstrated moringa extract suppression of ROS, preservation of cytochrome oxidase activity, and reduction in caspase production. Moringa extract demonstrated potent antioxidant properties with significant protection against gentamicin ototoxicity in cochlear explants.

Pioglitazone Ameliorates Gentamicin Ototoxicity by Affecting the TLR and STAT Pathways in the Early Postnatal Organ of Corti.

Noise trauma, infection, and ototoxic drugs are frequent external causes of hearing loss. With no pharmacological treatments currently available, understanding the mechanisms and pathways leading to auditory hair cell (HC) damage and repair is crucial for identifying potential pharmacological targets. Prior research has implicated increased reactive oxygen species (ROS) and inflammation as general mechanisms of hearing loss common to diverse causes. Novel targets of these two key mechanisms of auditory damage may provide new paths toward the prevention and treatment of hearing loss. Pioglitazone, an oral antidiabetic drug from the class of thiazolidinediones, acts as an agonist of the peroxisome proliferator-activated receptor-gamma (PPAR-γ) and is involved in the regulation of lipid and glucose metabolism. PPAR-γ is an important player in repressing the expression of inflammatory cytokines and signaling molecules. We evaluated the effects of pioglitazone in the mouse Organ of Corti (OC) explants to characterize its influence on signaling pathways involved in auditory HC damage. The OC explants was cultured with pioglitazone, gentamicin, or a combination of both agents. Pioglitazone treatment resulted in significant repression of interferon (IFN)-α and -gamma pathways and downstream cytokines, as assessed by RNA sequencing and quantitative PCR gene expression assays. More detailed investigation at the single gene and protein level showed that pioglitazone mediated its anti-inflammatory effects through alterations of the Toll-like receptor (TLR) and STAT pathways. Together, these results indicate that pioglitazone significantly represses IFN and TLR in the cochlea, dampening the activity of gentamicin-induced pathways. These data support our previous results demonstrating significant protection of auditory HCs in the OC explants exposed to pioglitazone and other PPAR-targeted agents.

Evaluating the Efficacy of Taurodeoxycholic Acid in Providing Otoprotection Using an in vitro Model of Electrode Insertion Trauma.

Cochlear implants (CIs) are widely used to provide auditory rehabilitation to individuals having severe to profound sensorineural hearing loss (SNHL). However, insertion of electrode leads to inner trauma and activation of inflammatory and apoptotic signaling cascades resulting in loss of residual hearing in implanted individuals. Pharmaceutical interventions that can target these signaling cascades hold great potential for preserving residual hearing by preventing sensory cell damage. Bile salts have shown efficacy in various regions of the body as powerful antioxidants and anti-inflammatory agents. However, their efficacy against inner ear trauma has never been explored. The objective of this study was to determine whether taurodeoxycholic acid (TDCA), a bile salt derivative, can prevent sensory cell damage employing an in vitro model of electrode insertion trauma (EIT). The organ of Corti (OC) explants were dissected from postnatal day 3 (P-3) rats and placed in serum-free media. Explants were divided into control and experimental groups: (1) untreated controls; (2) EIT; (3) EIT+ TDCA (different concentrations). Hair cell (HC) density, analyses of apoptosis pathway (cleaved caspase 3), levels of reactive oxygen species (ROS) as well as inducible nitric oxide synthase (iNOS) activity and Mitochondrial Membrane Potential (MMP) were assayed. Treatment with TDCA provided significant otoprotection against HC loss in a dose-dependent manner. The molecular mechanisms underlying otoprotection involved decreasing oxidative stress, lowering levels of iNOS, and abrogating generation of cleaved caspase 3. The results of the present study suggest that TDCA provides efficient otoprotection against EIT, in vitro and should be explored for developing pharmaceutical interventions to preserve residual hearing post-cochlear implantation.

Differential Effects of Low- and High-Dose Dexamethasone on Electrically Induced Damage of the Cultured Organ of Corti

An increased number of patients with residual hearing are undergoing cochlear implantation. A subset of these experience delayed hearing loss post-implantation, and the aetiology of this loss is not well understood. Our previous studies suggest that electrical stimulation can induce damage to hair cells in organ of Corti (OC) organotypic cultures. Dexamethasone has the potential to protect residual hearing due to its multiple effects on cells and tissue (e.g., anti-inflammatory, free radical scavenger). We therefore hypothesized that dexamethasone treatment could prevent electrical stimulation induced changes in the OC. Organ of Corti explants from neonatal rats (P2–4) were cultured for 24 h with two different concentrations of dexamethasone. Thereafter, OC were subjected to a charge-balanced biphasic pulsed electrical stimulation (0.44–2 mA) for a further 24 h. Unstimulated dexamethasone-treated OC served as controls. Outcome analysis included immunohistochemical labelling of ribbon synapses, histochemical analysis of free reactive oxygen species and morphological analysis of stereocilia bundles. Overall, the protective effects of dexamethasone on electrically induced damage in cochlear explants were moderate. High-dose dexamethasone protected bundle integrity at higher current levels. Low-dose dexamethasone tended to increase ribbon density in the apical region.

Cytokinesis and postabscission midbody remnants are regulated during mammalian brain development

Building a brain of the proper size and structure requires neural stem cells (NSCs) to divide with tight temporal and spatial control to produce different daughter cell types in proper numbers and sequence. Mammalian NSCs in the embryonic cortex must maintain their polarized epithelial structure as they undergo both early proliferative divisions and later neurogenic divisions. To do this, they undergo a polarized form of cytokinesis at the apical membrane that is not well understood. Here, we investigate whether polarized furrowing and abscission in mouse NSCs are regulated differently at earlier and later stages and in a cytokinesis mutant, Kif20b This mutant was previously shown to have microcephaly and elevated apoptosis of NSCs. We developed methods to live image furrow ingression and midbody abscission in NSCs within cortical explants. We find that polarized furrow ingression occurs at a steady rate and completes in ∼15 min at two different ages. However, ingression is slower in a subset of Kif20b mutant NSCs. Abscission is usually observed on both sides of the midbody and takes 65 to 75 min to complete. Surprisingly, abscission is accelerated in the Kif20b mutant NSCs. Postabscission midbody remnants are observed at the apical membranes of daughter cells and are much more abundant in early-stage cortices. After NSC divisions in vitro, midbody remnants are more often retained on the daughter cells of early proliferative divisions. Altogether, these results suggest that regulation of abscission timing and midbody remnants in embryonic NSCs may influence proper brain growth and structure.

Evaluating the Efficacy of L-N-acetylcysteine and Dexamethasone in Combination to Provide Otoprotection for Electrode Insertion Trauma.

Background: Electrode insertion trauma (EIT) during cochlear implantation (CI) can cause loss of residual hearing. L-N-acetylcysteine (L-NAC) and dexamethasone (Dex) have been individually shown to provide otoprotection albeit at higher concentrations that may be associated with adverse effects. Objective/Aims: The aim of this study is to determine whether L-NAC and Dex could be combined to decrease their effective dosage. Materials and Methods: The organ of Corti (OC) explants were divided into various groups: 1) control; 2) EIT; 3) EIT treated with different concentrations of Dex; 4) EIT treated with different concentrations of L-NAC; 5) EIT treated with L-NAC and Dex in combination. Hair cell (HC) density, levels of oxidative stress, proinflammatory cytokines and nitric oxide (NO) was determined. Results: There was a significant loss of HCs in explants subjected to EIT compared to the control group. L-NAC and Dex in combination was able to provide significant otoprotection at lower concentrations compared to individual drugs. Conclusions and Significance: A combination containing L-NAC and Dex is effective in protecting sensory cells at lower protective doses than each compound separately. These compounds can be combined allowing a decrease of potential side effects of each compound and providing significant otoprotection for EIT.

Ketamine decreases cell viability of bone explants and impairs bone healing in rats

BackgroundKetamine is a widely used anesthetic in experimental medicine. We have also used ketamine for surgical interventions and imaging in rats and found significantly impaired ossification between identically performed experiments, which only differed in the number of anesthetic events. In order to investigate this phenomenon, we estimated the absorbed ionizing radiation and also studied whether ketamine administration has disadvantageous effect on bone cell viability.MethodsSpongious bone chips and parietal bone disks were harvested from rats. Explants were incubated in stem cell media containing 0.02, 0.2 and 2 mM ketamine. After 3 days of incubation, tetrazolium-based spectrophotometric assay was performed to measure cell viability. Size-specific dose estimation was used to calculate ionizing radiation of computed tomography imaging.ResultsWe found that ketamine supplementation with 0.2 mM slightly decreased cell viability, while 2 mM caused significant reduction both in the spongious and cortical explants. The cumulative ionizing radiation was found to be negligible compared to irradiation dosages used to impair ossification.ConclusionsWe conclude that multiple ketamine administration was responsible for the diminished regenerative potential of bone tissue in the present experimental setup. For this reason, we suggest that ketamine anesthesia should be avoided in studies investigating bone regeneration.

Dextran Labeling and Uptake in Live and Functional Murine Cochlear Hair Cells.

The hair cell mechanotransduction (MET) channel plays an important role in hearing. However, the molecular identity and structural information of MET remain unknown. Electrophysiological studies of hair cells revealed that the MET channel has a large conductance and is permeable to relatively large fluorescent cationic molecules, including some styryl dyes and Texas Red-labeled aminoglycoside antibiotics. In this protocol, we describe a method to visualize and evaluate the uptake of fluorescent dextrans in hair cells of the organ of Corti explants that can be used to assay for functional MET channels. We found that 3 kDa Texas Red-labeled dextran specifically labels functional auditory hair cells after 1-2 h incubation. In particular, 3 kDa dextran labels the two shorter stereocilia rows and accumulates in the cell body in a diffuse pattern when functional MET channels are present. An additional vesicle-like pattern of labeling was observed in the cell body of hair cells and surrounding supporting cells. Our data suggest that 3 kDa Texas-Red dextran can be used to visualize and study two pathways for cellular dye uptake; a hair cell-specific entry route through functional MET channels and endocytosis, a pattern also available to larger dextran.

Hyaluronidase reduced edema after experimental traumatic brain injury

Cerebral edema and the subsequent increased intracranial pressure are associated with mortality and poor outcome following traumatic brain injury. Previous in vitro studies have shown that the Gibbs-Donnan effect, which describes the tendency of a porous, negatively charged matrix to attract positive ions and water, applies to brain tissue and that enzymatic reduction of the fixed charge density can prevent tissue swelling. We tested whether hyaluronidase, an enzyme that degrades the large, negatively charged glycosaminoglycan hyaluronan, could reduce brain edema after traumatic brain injury. In vivo, intracerebroventricular injection of hyaluronidase after controlled cortical impact in mice reduced edema in the ipsilateral hippocampus at 24 h by both the wet-weight/dry-weight method (78.15 ± 0.65% vs. 80.4 ± 0.46%; p < 0.01) and T2-weighted magnetic resonance imaging (13.88 ± 3.09% vs. 29.23 ± 6.14%; p < 0.01). Hyaluronidase did not adversely affect blood–brain-barrier-integrity measured by dynamic contrast-enhanced magnetic resonance imaging, nor did hyaluronidase negatively affect functional recovery after controlled cortical impact measured with the rotarod or Morris water maze tasks. Reduction of fixed charge density by hyaluronidase was confirmed in cortical explants in vitro (5.46 ± 1.15 µg/mg vs. 7.76 ± 1.87 µg/mg; p < 0.05). These data demonstrate that targeting the fixed charge density with hyaluronidase reduced edema in an in vivo mouse model of traumatic brain injury.

Single neurons activate cortical networks

Neuroscience All living reptiles and mammals have a brain cortex derived from a common primordial cortex. Thus, they may retain traces of this common ancestry in their structure, connectivity, and function. Hemberger et al. simultaneously sampled action potentials from hundreds of neurons in intact turtle cortical explants and found that minimal manipulation of single cortical pyramidal cells had detectable effects on other neurons and on the animal's behavior. With only one to three spikes, single pyramidal neurons could trigger distributed patterns of firing that cascaded across neurons, space, and time in the form of reliable firing sequences. These sequences are similar to those produced during replay events in the mammalian hippocampus. These data provide evidence for chained synaptic activation in a cortical structure supporting the synfire chain hypothesis. Neuron 10.1016/j.neuron.2019.07.017 (2019).