Laura J. Smithson

Technical Strategies to Isolate Olfactory Ensheathing Cells for Intraspinal Implantation

Over the past few years, the idea of using intraspinal implantations of olfactory ensheathing cells (OECs) as a therapeutic strategy to enhance recovery after spinal cord injury has quickly moved from experimentation with laboratory mammals to surgical approaches for paralyzed humans. Despite this progression, several important issues have yet to be thoroughly addressed: for instance, which of the many methods currently being used best yields enriched populations of OECs, and how such purity can be empirically tested and validated among different mammalian species, including humans. Here we offer an authoritative review of those methods used to isolate OECs from the olfactory mucosa and/or olfactory bulbs of rats, mice, dogs, pigs, non-human primates, and humans. As well, we assess which biomarkers are currently being utilized to determine the relative proportions of OECs and contaminating cells in these glial cultures. Although there have been numerous review articles regarding OECs in vitro, our review is unique in that it offers a critical assessment of the methods currently being used to generate cultures of mammalian OECs. More specifically, we examine the issue of culture contamination by phenotypically similar Schwann cells. This review is timely because recent clinical usage of OECs has come under intense criticism for a number of reasons, including the reliable identification of cultured human OECs. We believe that once these methodological issues of isolation and characterization of OECs have been resolved, this glial population will offer paralyzed individuals a truly viable cellular strategy for intraspinal therapy.

A comparative examination of biomarkers for olfactory ensheathing cells in cats and guinea pigs.

We investigated the neurochemical characteristics of olfactory ensheathing cells (OECs) in adult cats and in adult guinea pigs. Three conventional biomarkers for OECs, p75 neurotrophin receptor (p75NTR), S100, and glial fibrillary acidic protein (GFAP), as well as two recently identified biomarkers, smooth muscle alpha-actin (SMA) and calponin, were used. We found that 1) antibodies against SMA and S100 yielded positive immunostaining of mucosal and bulbar OECs in cats and guinea pigs; 2) antibodies against GFAP gave positive immunostaining of mucosal and bulbar OECs in cats; and 3) antibodies against calponin yielded positive immunostaining of bulbar OECs in adult cats. Unexpectedly, antibodies against p75NTR failed to positively stain mucosal and bulbar OECs in cats and guinea pigs, and antibodies against GFAP and calponin failed to positively stain mucosal and bulbar OECs in guinea pigs. These findings show the importance for empirical testing of all biomarkers for OECs among different mammalian species when attempting to identify these cells in vivo, in vitro, and following intraspinal implantation.

Nerve Growth Factor Promoter Activity Revealed in Mice Expressing Enhanced Green Fluorescent Protein

Nerve growth factor (NGF) and its precursor proNGF are perhaps the best described growth factors of the mammalian nervous system. There remains, however, a paucity of information regarding the precise cellular sites of proNGF/NGF synthesis. Here we report the generation of transgenic mice in which the NGF promoter controls the ectopic synthesis of enhanced green fluorescent protein (EGFP). These transgenic mice provide an unprecedented resolution of both neural cells (e.g., neocortical and hippocampal neurons) and non‐neural cells (e.g., renal interstitial cells and thymic reticular cells) that display NGF promoter activity from postnatal development to adulthood. Moreover, the transgene is inducible by injury. At 2 days after sciatic nerve ligation, a robust population of EGFP‐positive cells is seen in the proximal nerve stump. These transgenic mice offer novel insights into the cellular sites of NGF promoter activity and can be used as models for investigating the regulation of proNGF/NGF expression after injury. J. Comp. Neurol. 519:2522–2545, 2011.

Microglial/macrophage cells in mammalian olfactory nerve fascicles

This is the first description of a population of Iba1‐ and annexin A3‐immunopositive cells residing in the peripheral olfactory nerves of adult rats and adult cats. Based on their ramified appearance, positive immunostaining for the monocytic markers Iba1 and annexin A3, and reactivity to bulbectomy (in adult rats), these cells found within the olfactory nerve fascicles of both mammalian species meet several important criteria for their designation as microglia/macrophages. These Iba1‐/annexin A3‐immunopositive cells may be uniquely positioned to protect against the potential spread of dangerous environmental xenobiotics (such as viruses and toxins) into the brain, where such pathogens may contribute to the development of neurological diseases, such Alzheimers and Parkinsons diseases.

Reactive astrocytes associated with plaques in TgCRND8 mouse brain and in human Alzheimer brain express phosphoprotein enriched in astrocytes (PEA-15)

To identify potential biomarkers associated with Alzheimers disease (AD)‐like neuropathologies in the murine brain, we conducted proteomic analyses of neocortices from TgCRND8 mice. Here we found that phosphoprotein enriched in astrocytes 15 kDa (PEA‐15) is expressed at higher levels in the neocortical proteomes from 6‐month old TgCRND8 mice, as compared to non‐transgenic mice. Immunostaining for PEA‐15 revealed reactive astrocytes associated with the neocortical amyloid plaques in TgCRND8 mice and in post‐mortem human AD brains. This is the first report of increased phosphoprotein enriched in astrocytes (PEA‐15) expression in reactive astrocytes of an AD mouse model and human AD brains.

Olfactory ensheathing cells of hamsters, rabbits, monkeys, and mice express α-smooth muscle actin

Olfactory ensheathing cells (OECs) are the chief glial population of the mammalian olfactory nervous system, residing in the olfactory mucosa and at the surface of the olfactory bulb. We investigated the neurochemical features of OECs in a variety of mammalian species (including adult hamsters, rabbits, monkeys, and mice, as well as fetal pigs) using three biomarkers: α-smooth muscle actin (αSMA), S100β, and glial fibrillary acidic protein (GFAP). Mucosal and bulbar OECs from all five mammalian species express S100β. Both mucosal and bulbar OECs of monkeys express αSMA, yet only bulbar OECs of hamsters and only mucosal OECs of rabbits express αSMA as well. Mucosal OECs, but not bulbar OECs, also express GFAP in hamsters and monkeys; mice, by comparison, have only a sparse population of OECs expressing GFAP. Though αSMA immunostaining is not detected in OECs of adult mice, GFAP-expressing mucosal OECs isolated from adult mice do coexpress αSMA in vitro. Moreover, mucosal OECs from adult mutant mice lacking αSMA expression display perturbed cellular morphology (i.e., fewer cytoplasmic processes extending among the hundreds of olfactory axons in the olfactory nerve fascicles and nuclei having degenerative features). In sum, these findings highlight the efficacy of αSMA and S100β as biomarkers of OECs from a variety of mammalian species. These observations provide definitive evidence that mammalian OECs express the structural protein αSMA (at various levels of detection), which appears to play a pivotal role in their ensheathment of olfactory axons.

Overexpression of nerve growth factor by murine smooth muscle cells: Role of the p75 neurotrophin receptor on sympathetic and sensory sprouting

Elevating levels of nerve growth factor (NGF) can have pronounced effects on the survival and maintenance of distinct populations of neurons. We have generated a line of transgenic mice in which NGF is expressed under the control of the smooth muscle α‐actin promoter. These transgenic mice have augmented levels of NGF protein in the descending colon and urinary bladder, so these tissues display increased densities of NGF‐sensitive sympathetic efferents and sensory afferents. Here we provide a thorough examination of sympathetic and sensory axonal densities in the descending colon and urinary bladder of NGF transgenic mice with and without the expression of the p75 neurotrophin receptor (p75NTR). In response to elevated NGF levels, sympathetic axons (immunostained for tyrosine hydroxylase) undergo robust collateral sprouting in the descending colon and urinary bladder of adult transgenic mice (i.e., those tissues having smooth muscle cells); this sprouting is not augmented in the absence of p75NTR expression. As for sensory axons (immunostained for calcitonin gene‐related peptide) in the urinary bladders of transgenic mice, fibers undergo sprouting that is further increased in the absence of p75NTR expression. Sympathetic axons are also seen invading the sensory ganglia of transgenic mice; these fibers form perineuronal plexi around a subpopulation of sensory somata. Our results reveal that elevated levels of NGF in target tissues stimulate sympathetic and sensory axonal sprouting and that an absence of p75NTR by sensory afferents (but not by sympathetic efferents) leads to a further increase of terminal arborization in certain NGF‐rich peripheral tissues. J. Comp. Neurol. 521:2621–2643, 2013.