Long-term viable chimeric nephrons generated from progenitor cells are a reliable model in cisplatin-induced toxicity.

Kidney organoids have shown promise as evaluation tools, but their in vitro maturity remains limited. Transplantation into adult mice has aided in maturation; however, their lack of urinary tract connection limits long-term viability. Thus, long-term viable generated nephrons have not been demonstrated. In this study, we present an approachable method in which mouse and rat renal progenitor cells are injected into the developing kidneys of neonatal mice, resulting in the generation of chimeric nephrons integrated with the host urinary tracts. These chimeric nephrons exhibit similar maturation to the host nephrons, long-term viability with excretion and reabsorption functions, and cisplatin-induced renal injury in both acute and chronic phases, as confirmed by single-cell RNA-sequencing. Additionally, induced human nephron progenitor cells differentiate into nephrons within the neonatal kidneys. Collectively, neonatal injection represents a promising approach for in vivo nephron generation, with potential applications in kidney regeneration, drug screening, and pathological analysis.

Detailed anatomic description of the lateral, transzygomatic approach to the middle fossa and rostral brainstem and its use in three dogs.

OBJECTIVE: To describe a craniectomy using a lateral, transzygomatic approach to the middle fossa and rostral brainstem, and to report clinical outcomes and complications in three dogs. ANIMALS: Two cadaver dogs and three client-owned dogs. Two of the client-owned dogs with middle fossa lesions, and one with a rostral brainstem lesion. METHODS: Two cadavers were used to describe the lateral, transzygomatic surgical approach to the middle fossa and rostral brainstem. The medical records of three dogs undergoing this surgical approach were reviewed for data on signalment, preoperative and postoperative neurological status, diagnostic imaging, surgical technique, complications, and outcome. RESULTS: Indications for this surgical approach included incisional biopsy (n = 1) and debulking surgery for brain lesions (n = 2). Definitive diagnoses were achieved in two cases, and tumor volume reduction in all cases. Two of the three dogs developed postoperative facial nerve paralysis ipsilateral to the surgical site, which resolved within 2 and 12 weeks after surgery. CONCLUSION: The lateral, transzygomatic approach provided useful access to ventrally located cerebral/skull base lesions in dogs without major complications.

What Is Your Diagnosis?

In collaboration with the American College of Veterinary Radiology.

Repeated administration of the NSAID meloxicam alters the plasma and urine lipidome.

Non-steroidal anti-inflammatories (NSAIDs), such as meloxicam, are the mainstay for treating painful and inflammatory conditions in animals and humans; however, the repeated administration of NSAIDs can cause adverse effects, limiting the long-term administration of these drugs to some patients. The primary aim of this study was to determine the effects of repeated meloxicam administration on the feline plasma and urine lipidome. Cats (n = 12) were treated subcutaneously with either saline solution or 0.3 mg/kg body weight of meloxicam daily for up to 31 days. Plasma and urine lipidome were determined by LC-MS before the first treatment and at 4, 9 and 13 and 17 days after the first administration of meloxicam. The repeated administration of meloxicam altered the feline plasma and urine lipidome as demonstrated by multivariate statistical analysis. The intensities of 94 out of 195 plasma lipids were altered by the repeated administration of meloxicam to cats (p < 0.05). Furthermore, we identified 12 lipids in plasma and 10 lipids in urine that could serve as biomarker candidates for discriminating animals receiving NSAIDs from healthy controls. Expanding our understanding about the effects of NSAIDs in the body could lead to the discovery of mechanism(s) associated with intolerance to NSAIDs.

Voltage-gated calcium and sodium channels mediate Sema3A retrograde signaling that regulates dendritic development.

Growing axons rely on local signaling at the growth cone for guidance cues. Semaphorin3A (Sema3A), a secreted repulsive axon guidance molecule, regulates synapse maturation and dendritic branching. We previously showed that local Sema3A signaling in the growth cones elicits retrograde retrograde signaling via PlexinA4 (PlexA4), one component of the Sema3A receptor, thereby regulating dendritic localization of AMPA receptor GluA2 and proper dendritic development. In present study, we found that nimodipine (voltage-gated L-type Ca(2+) channel blocker) and tetrodotoxin (TTX; voltage-gated Na(+) channel blocker) suppress Sema3A-induced dendritic localization of GluA2 and dendritic branch formation in cultured hippocampal neurons. The local application of nimodipine or TTX to distal axons suppresses retrograde transport of Venus-Sema3A that has been exogenously applied to the distal axons. Sema3A facilitates axonal transport of PlexA4, which is also suppressed in neurons treated with either TTX or nimodipine. These data suggest that voltage-gated calcium and sodium channels mediate Sema3A retrograde signaling that regulates dendritic GluA2 localization and branch formation.

Quantitative analysis of intraneuronal transport in human iPS neurons.

Induced pluripotent stem (iPS) cells are promising tools to investigate disease mechanism and develop new drugs. Intraneuronal transport, which is fundamental for neuronal survival and function, is vulnerable to various pharmacological and chemical agents and is disrupted in some neurodegenerative disorders. We applied a quantification method for axonal transport by counting CM-DiI-labeled particles traveling along the neurite, which allowed us to monitor and quantitate, for the first time, intraneuronal transport in human neurons differentiated from iPS cells (iCell neurons). We evaluated the acute effects of several anti-neoplastic agents that have been previously shown to affect intraneuronal transport. Vincristine, paclitaxel and oxaliplatin decreased the number of moving particle along neurites. Cisplatin, however, produced no effect on intraneuronal transport, which is in contrast to our previous report indicating that it inhibits transport in chick dorsal root ganglion neurons. Our system may be a useful method for assessing intraneuronal transport and neurotoxicity in human iPS neurons.

Cardiovascular actions of DOPA mediated by the gene product of ocular albinism 1.

l-3,4-Dihydroxyphenylalanine (DOPA) is the metabolic precursor of dopamine, and the single most effective agent in the treatment of Parkinson's disease. One problem with DOPA therapy for Parkinson's disease is its cardiovascular side effects including hypotension and syncope, the underlying mechanisms of which are largely unknown. We proposed that DOPA is a neurotransmitter in the central nervous system, but specific receptors for DOPA had not been identified. Recently, the gene product of ocular albinism 1 (OA1) was shown to possess DOPA-binding activity. It was unknown, however, whether or not OA1 is responsible for the actions of DOPA itself. Immunohistochemical examination revealed that OA1 was expressed in the nucleus tractus solitarii (NTS). OA1-positive cells adjacent to tyrosine hydroxylase-positive cell bodies and nerve fibers were detected in the depressor sites of the NTS. OA1 knockdown using oa1-specific shRNA-adenovirus vectors in the NTS reduced the expression levels of OA1 in the NTS. The prior injection of the shRNA against OA1 suppressed the depressor and bradycardic responses to DOPA but not to glutamate in the NTS of anesthetized rats. Thus OA-1 is a functional receptor of DOPA in the NTS, which warrants reexamination of the mechanisms for the therapeutic and untoward actions of DOPA.

Localization of ocular albinism-1 gene product GPR143 in the rat central nervous system.

L-3,4-Dihydroxyphenylalanine (DOPA) has been believed to be a precursor of dopamine, and itself being an inert amino acid. Previously, we have proposed DOPA as a neurotransmitter candidate in the central nervous system (CNS). Recent findings have suggested DOPA as an endogenous agonist of a G-protein coupled receptor, ocular albinism 1 gene product (OA1), which is highly expressed in the retinal pigmental epithelium. However, whether OA1 functions as a receptor for DOPA in vivo, and whether this receptor-ligand interaction is responsible for a wide variety of DOPA actions have not been determined yet. To gain insight into the functional implication of OA1, we perform immunohistochemical examination with anti-OA1 antibody to localize OA1 in the adult rat brain. We observed OA1 immunoreactive cells in the hippocampus, cerebral cortex, cerebellum cortex, striatum, substantia nigra, hypothalamic median eminence and supraoptic nucleus, nucleus tractus solitarii and caudal ventrolateral medulla and rostral ventrolateral medulla, medial habenular nucleus and olfactory bulb. This study reveals, for the first time, the unique distribution pattern of OA1-immunoreactive neurons and/or cells in the rat CNS.

Plexin-A4-dependent retrograde semaphorin 3A signalling regulates the dendritic localization of GluA2-containing AMPA receptors.

The dendritic targeting of neurotransmitter receptors is vital for dendritic development and function. However, how such localization is established remains unclear. Here we show that semaphorin 3A (Sema3A) signalling at the axonal growth cone is propagated towards the cell body by retrograde axonal transport and drives AMPA receptor GluA2 to the distal dendrites, which regulates dendritic development. Sema3A enhances glutamate receptor interacting protein 1-dependent localization of GluA2 in dendrites, which is blocked by knockdown of cytoplasmic dynein heavy chain. PlexinA (PlexA), a receptor component for Sema3A, interacts with GluA2 at the immunoglobulin-like Plexin-transcription-factor domain (PlexA-IPT) in somatodendritic regions. Overexpression of PlexA-IPT suppresses dendritic localization of GluA2 and induces aproximal bifurcation phenotype in the apical dendrites of CA1 hippocampal neurons. Thus, we propose a control mechanism by which retrograde Sema3A signalling regulates the glutamate receptor localization through trafficking of cis-interacting PlexA with GluA2 along dendrites.

Draxin, an axon guidance protein, affects chick trunk neural crest migration.

The neural crest is a multipotent population of migratory cells that arises in the central nervous system and subsequently migrates along defined stereotypic pathways. In the present work, we analyzed the role of a repulsive axon guidance protein, draxin, in the migration of neural crest cells. Draxin is expressed in the roof plate of the chick trunk spinal cord and around the early migration pathway of neural crest cells. Draxin modulates chick neural crest cell migration in vitro by reducing the polarization of these cells. When exposed to draxin, the velocity of migrating neural crest cells was reduced, and the cells changed direction so frequently that the net migration distance was also reduced. Overexpression of draxin also caused some early migrating neural crest cells to change direction to the dorsolateral pathway in the chick trunk region, presumably due to draxin's inhibitory activity. These results demonstrate that draxin, an axon guidance protein, can also affect trunk neural crest migration in the chick embryo.

Analysis of a repulsive axon guidance molecule, draxin, on ventrally directed axon projection in chick early embryonic midbrain.

The mesencephalic V neurons and tectobulbar axons in chick embryo project over long distances that appear during the early development of the chick optic tectum. The mesencephalic V neuron and tectobulbar axonal growth begin at Hamburger and Hamilton stage 14 and stage 18, respectively. Both fibers proceed downward from the dorsal to the ventral side of the lateral wall of the optic tectum and then turn caudally and join the medial longitudinal fasciculus. Their axons appear in the most superficial layer of the tectum at early stages and do not cross the dorsal midline of the tectum. Here, we report the role of draxin, a recently identified axon guidance protein, in the formation of the ventrally directed tectum axonal tracts in chicken embryo. draxin is expressed in a high dorsal to low ventral gradient in chick optic tectum. In vitro experiments show that draxin repels neurite outgrowth from dorsal tectum explants. In vivo overexpression resulted in inhibition or misrouting of axon growth in the tectum. Therefore, draxin may be an important member of the collection of repulsive guidance molecules that regulate the formation of the ventrally directed tectum axon tracts.

Draxin, a repulsive guidance protein for spinal cord and forebrain commissures.

Axon guidance proteins are critical for the correct wiring of the nervous system during development. Several axon guidance cues and their family members have been well characterized. More unidentified axon guidance cues are assumed to participate in the formation of the extremely complex nervous system. We identified a secreted protein, draxin, that shares no homology with known guidance cues. Draxin inhibited or repelled neurite outgrowth from dorsal spinal cord and cortical explants in vitro. Ectopically expressed draxin inhibited growth or caused misrouting of chick spinal cord commissural axons in vivo. draxin knockout mice showed defasciculation of spinal cord commissural axons and absence of all forebrain commissures. Thus, draxin is a previously unknown chemorepulsive axon guidance molecule required for the development of spinal cord and forebrain commissures.

Perforating ocular injury by Taser.

This report describes the features, treatment and outcome of globe perforation by a Taser dart electrode in a 21-year-old man. The Taser electrode caused mechanical iris, lens and retinal injury and consequent retinal detachment as result of proliferative vitreoretinopathy. The effect of electrical stimulation on ocular tissues is unknown. After the scleral and corneal wounds, traumatic cataract and retinal tear were repaired, the patient regained a visual acuity of 6/18. Nine months later a retinal detachment with proliferative vitreoretinopathy was discovered. The Taser may cause globe perforation and posterior segment injury. Understanding the barbed configuration of the dart electrode is important when extricating this device. Visual recovery is possible despite electric discharge of the Taser and suggests that the mechanism of ocular injury is largely mechanical.

Corneal transduction to inhibit angiogenesis and graft failure.

PURPOSE: To test whether lentivirus-mediated expression of an endostatin::kringle-5 (E::K-5) fusion gene has an inhibitory effect on neovascularization and failure of corneal transplants. METHODS: A lentiviral vector containing a fusion transgene comprising the human endostatin gene and the kringle-5 domain of the human plasminogen gene (E::K-5) was used for transduction of corneal buttons ex vivo. The corneal buttons were transplanted after overnight incubation in media containing either lentivirus or PBS. Sixteen rabbits underwent allogenic penetrating keratoplasty in one eye. The area of neovascularization from the limbus to within the graft was documented after surgery. RT-PCR was performed to demonstrate the presence of transgene mRNA within the graft. Histopathology was used to analyze neovascularization, inflammation, and rejection morphology. RESULTS: Less neovascularization was observed in corneas treated with the lentivirus E::K-5 fusion vector. Early onset and profound neovascularization was observed in control eyes. E::K-5-treated animals did not have graft failure, whereas five of the six control animals had graft failure, as classified by opacification of the graft. All E::K-5 transduced corneas tested were positive by RT-PCR for the unique fusion gene sequence. Histopathology corroborated a significant increase of blood vessel presence and inflammatory reaction in control compared with treated eyes. CONCLUSIONS: Corneas transduced with a lentivirus containing an endostatin::kringle-5 fusion gene demonstrated an inhibition of neovascularization and graft failure. E::K-5 gene transduction through a lentiviral vector system may be a useful adjunct to prevent graft neovascularization and corneal graft rejection in high-risk corneal transplants with antecedent rejection or neovascularization.