Brenton Luke Cavanagh

Thymidine Analogues for Tracking DNA Synthesis

Replicating cells undergo DNA synthesis in the highly regulated, S-phase of the cell cycle. Analogues of the pyrimidine deoxynucleoside thymidine may be inserted into replicating DNA, effectively tagging dividing cells allowing their characterisation. Tritiated thymidine, targeted using autoradiography was technically demanding and superseded by 5-bromo-2-deoxyuridine (BrdU) and related halogenated analogues, detected using antibodies. Their detection required the denaturation of DNA, often constraining the outcome of investigations. Despite these limitations BrdU alone has been used to target newly synthesised DNA in over 20,000 reviewed biomedical studies. A recent breakthrough in “tagging DNA synthesis” is the thymidine analogue 5-ethynyl-2′-deoxyuridine (EdU). The alkyne group in EdU is readily detected using a fluorescent azide probe and copper catalysis using ‘Huisgen’s reaction’ (1,3-dipolar cycloaddition or ‘click chemistry’). This rapid, two-step biolabelling approach allows the tagging and imaging of DNA within cells whilst preserving the structural and molecular integrity of the cells. The bio-orthogonal detection of EdU allows its application in more experimental assays than previously possible with other “unnatural bases”. These include physiological, anatomical and molecular biological experimentation in multiple fields including, stem cell research, cancer biology, and parasitology. The full potential of EdU and related molecules in biomedical research remains to be explored.

Olfactory stem cells can be induced to express chondrogenic phenotype in a rat intervertebral disc injury model.

BACKGROUND In humans, lower back pain is one of the most common causes of morbidity. Many studies implicate degeneration of intervertebral discs as the cause. In the normal intervertebral disc, the nucleus pulposus exerts a hydrostatic pressure against the constraining annulus fibrosus, which allows the disc to maintain flexibility between adjacent vertebrae, while absorbing necessary compressive forces. The nucleus pulposus performs this role because of its hydrophilic gel-like structure. The extracellular matrix of the nucleus pulposus is up to 80% hydrated, as a result of large amounts of the aggregating proteoglycan, chondroitin sulfate proteoglycan (CSPG). This proteoglycan is enmeshed in a randomly orientated network of fine collagen Type II (CT2) fibers. STUDY DESIGN AND PURPOSE: A useful adult tissue-derived stem cell is that from the olfactory mucosa, the organ of smell. These cells, accessible in humans from nasal biopsies, are multipotent and are able to make many cell types from all germ layers. They are easily grown in vitro and can be expanded to large numbers and stored frozen. These qualities indicate the potential for autologous transplantation for disc repair. In this article, using a rat model, we explore the hypothesis that olfactory stem cells can differentiate into a nucleus pulposus chondrocyte phenotype in vitro, as well as in vivo after transplantation into the injured intervertebral disc. PATIENT SAMPLE Female rats (14 weeks) were anesthetized with xylazine/ketamine. The abdominal wall was shaved and injected with local anesthetic (lidocaine) before incision. The ventral part of the lumbar spine, including two intervertebral discs, was exposed. Disc degeneration was then induced in the two exposed discs by needle aspiration of the nucleus pulposus. The prominent spina iliaca posterior superior was used as an anatomical landmark for identification of the first disc. Two weeks later, one injured intervertebral disc was exposed in a second, similar, surgery and 20,000 olfactory neurosphere-derived cells were transplanted with a 25-G needle. OUTCOME MEASURES In vitro induction of nucleus pulposus chondrocyte phenotype is measured by the percentage of cells expressing CT2 and CSPG. In vivo, a successful outcome is evidence of engraftment of donor-derived cells and their expression of CT2 and CSPG. METHODS In this article, we tested two hypotheses: the first that progenitor cells within olfactory neurospheres could be induced to express markers distinctive of the nucleus pulposus when placed in vitro in a coculture experiment. The second hypothesis tested the same induction in genetically labeled transplanted cells within damaged vertebral discs in vivo. The two markers measured are those held by current literature to engender the necessary cushioning characteristics of nucleus pulposus, CT2 and CSPG. RESULTS Our experiments demonstrated virtually 100% induction of these two markers in vitro. Also, this induction was achieved in donor-derived cells after delivery to the nucleus pulposus region of animals whose discs had previously been lesioned 2 weeks before transplant. CONCLUSIONS These results provide a rationale for moving toward more extensive larger animal studies for assessment of regeneration before human trials where relief of symptoms can be more easily assessed.

Automated classification of dopaminergic neurons in the rodent brain

Accurate morphological characterization of the multiple neuronal classes of the brain would facilitate the elucidation of brain function and the functional changes that underlie neurological disorders such as Parkinsons diseases or Schizophrenia. Manual morphological analysis is very time-consuming and suffers from a lack of accuracy because some cell characteristics are not readily quantified. This paper presents an investigation in automating the classification of dopaminergic neurons located in the brainstem of the rodent, a region critical to the regulation of motor behaviour and is implicated in multiple neurological disorders including Parkinsons disease. Using a Carl Zeiss Axioimager Z1 microscope with Apotome, salient information was obtained from images of dopaminergic neurons using a structural feature extraction technique. A data set of 100 images of neurons was generated and a set of 17 features was used to describe their morphology. In order to identify differences between neurons, 2-dimensional and 3-dimensional image representations were analyzed. This paper compares the performance of three popular classification methods in bioimage classification (Support Vector Machines (SVMs), Back Propagation Neural Networks (BPNNs) and Multinomial Logistic Regression (MLR)), and the results show a significant difference between machine classification (with 97% accuracy) and human expert based classification (72% accuracy).

A method for isolating cortical interneurons sharing the same birthdays for gene expression studies

Abstract The two neuronal populations in the cortex, pyramidal neurons and interneurons, can be separated based on neurotransmitter identity, however, within this segregation a large degree of diversity exists. Investigations into the molecular diversity of neurons are impeded by the inability to isolate cell populations born at different times for gene expression analysis. Developing interneurons may be distinguished by the expression of Glutamic Acid Decarboxylase‐67 (GAD67). Neuronal birthdating using nucleoside analogs is an effective means of identifying coetaneous interneurons. Using these two features, neurotransmitter identity and birthdating, we have developed a method to isolate migrating interneurons using fluorescent‐activated cell sorting (FACS) for RNA extraction and gene expression analysis. We utilized 5‐ethynyl‐2′‐deoxyuridine (EdU) to birthdate interneuron cohorts and the GAD67 knock‐in GFP transgenic mice to identify interneurons. In combination, we achieved simultaneous detection of GFP and EdU signals during FACS sorting of coetaneous interneurons with minimum loss of RNA integrity. RNA quality was deemed to be satisfactory by quantitative polymerase chain reaction (qPCR) for the interneuron‐specific transcript Gad67. HighlightsCortical interneurons sharing the same birthdates can be isolated by FACs sorting.Optimized EdU detection gives good signal and preserves cellular RNA integrity.PFA fixation improves concurrent GFP signal detection in two‐colour cell sorting of EdU‐labelled interneurons.RNA extracted from GFP+/EdU+ and GFP−/EdU+ cells can be used for qPCR analysis.

Olfactory ensheathing cells but not fibroblasts reduce the duration of autonomic dysreflexia in spinal cord injured rats

Autonomic dysreflexia is a common complication after high level spinal cord injury and can be life-threatening. We have previously shown that the acute transplantation of olfactory ensheathing cells into the lesion site of rats transected at the fourth thoracic spinal cord level reduced autonomic dysreflexia up to 8weeks after spinal cord injury. This beneficial effect was correlated with changes in the morphology of sympathetic preganglionic neurons despite the olfactory cells surviving no longer than 3weeks. Thus the transitory presence of olfactory ensheathing cells at the injury site initiated long-term functional as well as morphological changes in the sympathetic preganglionic neurons. The primary aim of the present study was to evaluate whether olfactory ensheathing cells survive after transplantation within the parenchyma close to sympathetic preganglionic neurons and whether, in this position, they still reduce the duration of autonomic dysreflexia and modulate sympathetic preganglionic neuron morphology. The second aim was to quantify the density of synapses on the somata of sympathetic preganglionic neurons with the hypothesis that the reduction of autonomic dysreflexia requires synaptic changes. As a third aim, we evaluated the cell type-specificity of olfactory ensheathing cells by comparing their effects with a control group transplanted with fibroblasts. Animals transplanted with OECs had a faster recovery from hypertension induced by colorectal distension at 6 and 7weeks but not at 8weeks after T4 spinal cord transection. Olfactory ensheathing cells survived for at least 8weeks and were observed adjacent to sympathetic preganglionic neurons whose overall number of primary dendrites was reduced and the synaptic density on the somata increased, both caudal to the lesion site. Our results showed a long term cell type-specific effects of olfactory ensheathing cells on sympathetic preganglionic neurons morphology and on the synaptic density on their somata, and a transient cell type-specific reduction of autonomic dysreflexia.

ISDN2012_0252Genesis and development of dopaminergic interneurons in the adult brain

and GABAergic neurons to identify alterations in network dynamics produced by TBI in comparison to sham injured mice. We used GAD67 knock-in mouse that expressed GFP in all GABAergic neurons. In these mice, we recorded from three groups of cells: excitatory neurons (EGFP-negative and SR101-negative), GABAergic neurons (EGFP-positive and SR101-negative), and astrocytes (EGFP-negative and SR101-positive) with the goal to characterize cell-type specific TBI-induced dysfunction. We compared parameters like resting potential, voltage-dependent whole-cell currents, spontaneous activity (EPSPs) and action potential characteristics between cells in the proximity of the injury and in similar regions of slices from sham-operated animals. Combined measurements of membrane potential and calcium activity were performed to further elucidate the mechanisms underlying calcium transients. Our results suggest desynchronization of neocortical activity following TBI with an impaired inhibition underling hyperactivity/hypoactivity of excitatory neurons possibly due to disruption of feedback inhibition. Taken together the capacity to simultaneously image calcium dynamics in three different cell types with high spatial and temporal resolution along with whole-cell recordings in individual neurons promises to serve as a valuable tool for a comprehensive and quantitative analysis of molecular mechanisms in barrel cortex and thalamocortical circuit dysfunction underlying the pathophysiology of TBI.

Induced nigrostriatal degeneration in rodents, as models for neuroprotective and regenerative strategies for Parkinson's disease

and GABAergic neurons to identify alterations in network dynamics produced by TBI in comparison to sham injured mice. We used GAD67 knock-in mouse that expressed GFP in all GABAergic neurons. In these mice, we recorded from three groups of cells: excitatory neurons (EGFP-negative and SR101-negative), GABAergic neurons (EGFP-positive and SR101-negative), and astrocytes (EGFP-negative and SR101-positive) with the goal to characterize cell-type specific TBI-induced dysfunction. We compared parameters like resting potential, voltage-dependent whole-cell currents, spontaneous activity (EPSPs) and action potential characteristics between cells in the proximity of the injury and in similar regions of slices from sham-operated animals. Combined measurements of membrane potential and calcium activity were performed to further elucidate the mechanisms underlying calcium transients. Our results suggest desynchronization of neocortical activity following TBI with an impaired inhibition underling hyperactivity/hypoactivity of excitatory neurons possibly due to disruption of feedback inhibition. Taken together the capacity to simultaneously image calcium dynamics in three different cell types with high spatial and temporal resolution along with whole-cell recordings in individual neurons promises to serve as a valuable tool for a comprehensive and quantitative analysis of molecular mechanisms in barrel cortex and thalamocortical circuit dysfunction underlying the pathophysiology of TBI.