Resilience in adolescence during the COVID-19 crisis in Canada.

BACKGROUND: The COVID-19 pandemic constitutes a social crisis that will have long-term health consequences for much of the global population, especially for adolescents. Adolescents are triply affected as they: 1) are experiencing its immediate, direct effects, 2) will carry forward health habits they develop now into adulthood, and 3) as future parents, will shape the early life health of the next generation. It is therefore imperative to assess how the pandemic is influencing adolescent wellbeing, identify sources of resilience, and outline strategies for attenuating its negative impacts. METHODS: We report the results of longitudinal analyses of qualitative data from 28 focus group discussions (FGDs) with 39 Canadian adolescents and of cross-sectional analyses of survey data from 482 Canadian adolescents gathered between September 2020 and August 2021. FGD participants and survey respondents reported on their: socio-demographic characteristics; mental health and wellbeing before and during the pandemic; pre- and during-pandemic health behaviours; experiences living through a crisis; current perceptions of their school, work, social, media, and governmental environments; and ideas about pandemic coping and mutual aid. We plotted themes emerging from FGDs along a pandemic timeline, noting socio-demographic variations. Following assessment for internal reliability and dimension reduction, quantitative health/wellbeing indicators were analyzed as functions of composite socio-demographic, health-behavioural, and health-environmental indicators. RESULTS: Our mixed methods analyses indicate that adolescents faced considerable mental and physical health challenges due to the pandemic, and were generally in poorer health than expected in non-crisis times. Nevertheless, some participants showed significantly better outcomes than others, specifically those who: got more exercise; slept better; were food secure; had clearer routines; spent more time in nature, deep in-person social relationships, and leisure; and spent less time on social media. CONCLUSIONS: Support for youth during times of crisis is essential to future population health because adolescence is a period in the life course which shapes the health behaviours, socio-economic capacities, and neurophysiology of these future parents/carers and leaders. Efforts to promote resilience in adolescents should leverage the factors identified above: helping them find structure and senses of purpose through strong social connections, well-supported work and leisure environments, and opportunities to engage with nature.

Addressing embodied inequities in health: how do we enable improvement in women's diet in pregnancy?

OBJECTIVE: To disrupt cycles of health inequity, traceable to dietary inequities in the earliest stages of life, public health interventions should target improving nutritional wellbeing in preconception/pregnancy environments. This requires a deep engagement with pregnant/postpartum people (PPP) and their communities (including their health and social care providers, HSCP). We sought to understand the factors that influence diet during pregnancy from the perspectives of PPP and HSCP, and to outline intervention priorities. DESIGN: We carried out thematic network analyses of transcripts from ten focus group discussions (FGD) and one stakeholder engagement meeting with PPP and HSCP in a Canadian city. Identified themes were developed into conceptual maps, highlighting local priorities for pregnancy nutrition and intervention development. SETTING: FGD and the stakeholder meeting were run in predominantly lower socioeconomic position (SEP) neighbourhoods in the sociodemographically diverse city of Hamilton, Canada. PARTICIPANTS: All local, comprising twenty-two lower SEP PPP and forty-three HSCP. RESULTS: Salient themes were resilience, resources, relationships and the embodied experience of pregnancy. Both PPP and HSCP underscored that socioeconomic-political forces operating at multiple levels largely determined the availability of individual and relational resources constraining diet during pregnancy. Intervention proposals focused on cultivating individual and community resilience to improve early-life nutritional environments. Participants called for better-integrated services, greater income supports and strengthened support programmes. CONCLUSIONS: Hamilton stakeholders foregrounded social determinants of inequity as main factors influencing pregnancy diet. They further indicated a need to develop interventions that build resilience and redistribute resources at multiple levels, from the household to the state.

Translating the Developmental Origins of Health and Disease concept to improve the nutritional environment for our next generations: a call for a reflexive, positive, multi-level approach.

Evidence supporting the Developmental Origins of Health and Disease (DOHaD) hypothesis indicates that improving early life environments can reduce non-communicable disease risks and improve health over the lifecourse. A widespread understanding of this evidence may help to reshape structures, guidelines and individual behaviors to better the developmental conditions for the next generations. Yet, few efforts have yet been made to translate the DOHaD concept beyond the research community. To understand why, and to identify priorities for DOHaD Knowledge Translation (KT) programs, we review here a portion of published descriptions of DOHaD KT efforts and critiques thereof. We focus on KT targeting people equipped to apply DOHaD knowledge to their everyday home or work lives. We identified 17 reports of direct-to-public DOHaD KT that met our inclusion criteria. Relevant KT programs have been or are being initiated in nine countries, most focusing on secondary school students or care-workers-in-training; few target parents-to-be. Early indicators suggest that such programs can empower participants. Main critiques of DOHaD KT suggest it may overburden mothers with responsibility for children's health and health environments, minimizing the roles of other people and institutions. Simultaneously, though, many mothers-to-be seek reliable guidance on prenatal health and nutrition, and would likely benefit from engagement with DOHaD KT. We thus recommend emphasizing solidarity, and bringing together people likely to one day become parents (youth), people planning pregnancies, expecting couples, care workers and policymakers into empowering conversation about DOHaD and about the importance and complexity of early life environments.

Rho signaling and axon regeneration.

Several major advances in our understanding of axon regeneration and functional repair in the central nervous system (CNS) together with new insights about molecular signaling pathways have led to development of viable drug candidates to treat spinal cord injury. In this review, we focus on Rho, an intracellular small GTPase that is part of a family of highly related proteins that are present in all cells as important signaling switches. Multiple lines of evidence have validated the Rho pathway as important in controlling axon growth and regeneration after neurotrauma in the CNS. The first part of this review will provide the evidence that Rho is a convergent point of signaling important for axon regeneration in the growth inhibitory environment of the adult CNS. The second part of the review will focus on efforts to target Rho to promote regeneration in vivo. The final section of the review will summarize clinical results with Cethrin, a Rho inhibitor that has completed phase I/IIa clinical testing.

Enhanced survival and regeneration of axotomized retinal neurons by repeated delivery of cell-permeable C3-like Rho antagonists.

Inactivation of Rho GTPase with a single intraocular injection of Rho antagonists stimulates survival and regeneration of retinal ganglion cells (RGCs) after optic nerve injury. However, this effect is short-lived. Here we tested the impact of multiple injections of C3-like Rho antagonists on RGC viability and axon regeneration after optic nerve lesion. Our data show that both neuronal survival and axon regeneration were enhanced with repeated delivery of cell-permeable C3. We found an approximately 1.5-fold increase in RCG survival when additional Rho antagonist injections were performed after the first week from the time of lesion. In contrast, increased regeneration required early inactivation of Rho and injections performed in the second week did not further enhance regenerative outcome. These results reveal differences in the length of the therapeutic windows through which Rho inactivation acts on RGC survival or regeneration after axotomy.

Expression of netrin-1 and its receptors DCC and UNC-5H2 after axotomy and during regeneration of adult rat retinal ganglion cells.

Netrins are a family of chemotropic factors that guide axon outgrowth during development; however, their function in the adult CNS remains to be established. We examined the expression of the netrin receptors DCC and UNC5H2 in adult rat retinal ganglion cells (RGCs) after grafting a peripheral nerve (PN) to the transected optic nerve and following optic nerve transection alone. In situ hybridization revealed that both Dcc and Unc5h2 mRNAs are expressed by normal adult RGCs. In addition, netrin-1 was found to be constitutively expressed by RGCs. Quantitative analysis using in situ hybridization demonstrated that both Dcc and Unc5h2 were down-regulated by RGCs following axotomy. In the presence of an attached PN graft, Dcc and Unc5h2 were similarly down-regulated in surviving RGCs regardless of their success in regenerating an axon. Northern blot analysis demonstrated expression of netrin-1 in both optic and sciatic nerve, and Western blot analysis revealed the presence of netrin protein in both nerves. Immunohistochemical analysis indicated that netrin protein was closely associated with glial cells in the optic nerve. These results suggest that netrin-1, DCC, and UNC5H2 may contribute to regulating the regenerative capacity of adult RGCs.

Spinal cord repair: strategies to promote axon regeneration.

Neurons in the central nervous system have a remarkable capacity to regenerate their transected axons when provided with an appropriate growth environment. Advances in our understanding of axon regeneration have allowed the development of different experimental strategies to stimulate axon regeneration in animal models of spinal cord injury. Growth inhibitory proteins block axon regeneration in the CNS, and many of these proteins have been identified. Various methods that are now used to stimulate regeneration in the injured spinal cord are directed at overcoming the growth inhibitory environment of the CNS. Three general approaches tested in vivo stimulate regeneration in the spinal cord. First, antibodies that bind inhibitory proteins in myelin allow axon regeneration in the CNS. Second, methods that modulate neuronal intracellular signaling allow axons to grow directly on the inhibitory substrate of the CNS. Third, transplantation of cells to the lesioned spinal cord promotes repair. In this paper we review current advances in each of these research domains.

Retinal ganglion cell and nonneuronal cell responses to a microcrush lesion of adult rat optic nerve.

Injury of the optic nerve has served as an important model for the study of cell death and axon regeneration in the CNS. Analysis of axon sprouting and regeneration after injury by anatomical tracing are aided by lesion models that produce a well-defined injury site. We report here the characterization of a microcrush lesion of the optic nerve made with 10-0 sutures to completely transect RGC axons. Following microcrush lesion, 62% of RGCs remained alive 1 week later, and 28% of RGCs, at 2 weeks. Optic nerve sections stained by hematoxylin-based methods showed a thin line of intensely stained cells that invaded the lesion site at 24 h after microcrush lesion. The lesion site became increasingly disorganized by 2 weeks after injury, and both macrophages and blood vessels invaded the lesion site. The microcrush lesion was immunoreactive for chondroitin sulfate proteoglycans (CSPG), and an adjacent GFAP-negative zone developed early after the lesion, disappearing by 1 week. Luxol fast blue staining showed a myelin-free zone at the lesion site, and myelin remained distal to the lesion at 8 weeks. To study the axonal response to microcrush lesion, anterograde tracing was used. Within 6 h after injury all RGC axons retracted back from the site of lesion. By 1 week after injury, axons regrew toward the lesion, but most stopped abruptly at the injury scar. The few axons that were able to cross the injury site did not extend further in the optic nerve white matter by 8 weeks postlesion. Our observations suggest that both the CSPG-positive scar and the myelin-derived growth inhibitory proteins contribute to the failure of RGC regeneration after injury.

A therapeutic vaccine approach to stimulate axon regeneration in the adult mammalian spinal cord.

Axon growth inhibitors associated with myelin play an important role in the failure of axon regeneration in the adult mammalian central nervous system (CNS). Several inhibitors are present in the mature CNS. We now present a novel therapeutic vaccine approach in which the animals' own immune system is stimulated to produce polyclonal antibodies that block myelin-associated inhibitors without producing any detrimental cellular inflammatory responses. Adult mice immunized in this manner showed extensive regeneration of large numbers of axons of the corticospinal tracts after dorsal hemisection of the spinal cord. The anatomical regeneration led to recovery of certain hind limb motor functions. Furthermore, antisera from immunized mice were able to block myelin-derived inhibitors and promote neurite growth on myelin in vitro.

Inactivation of Rho signaling pathway promotes CNS axon regeneration.

Regeneration in the CNS is blocked by many different growth inhibitory proteins. To foster regeneration, we have investigated a strategy to block the neuronal response to growth inhibitory signals. Here, we report that injured axons regrow directly on complex inhibitory substrates when Rho GTPase is inactivated. Treatment of PC12 cells with C3 enzyme to inactivate Rho and transfection with dominant negative Rho allowed neurite growth on inhibitory substrates. Primary retinal neurons treated with C3 extended neurites on myelin-associated glycoprotein and myelin substrates. To explore regeneration in vivo, we crushed optic nerves of adult rat. After C3 treatment, numerous cut axons traversed the lesion to regrow in the distal white matter of the optic nerve. These results indicate that targeting signaling mechanisms converging to Rho stimulates axon regeneration on inhibitory CNS substrates.

Organization of point contacts in neuronal growth cones.

Growth cones from rat dorsal root ganglia plated on laminin contain integrin clusters over the entire growth cone surface, and growth cones make transient adhesions at sites called point contacts. We examined, by immunocytochemistry and confocal microscopy, the composition and distribution of point contacts in neuronal growth cones. Vinculin was concentrated in the central domain of growth cones and at the tips of filopodia. Vinculin was specifically associated with integrin clusters at the membrane-substrate interface and thus marked point contacts. The cytoskeletal proteins paxillin and talin colocalized with beta1 integrin in a subpopulation of clusters restricted to the central domain of the growth cone and to the tips of filopodia. The neuron-specific kinase, FAK+ also distributed with the vinculin-positive clusters. The Rho family proteins RhoA, RhoB, and Cdc42 were present in growth cones, and a few Rho clusters were colocalized with vinculin. Examination of proteins resistant to detergent extraction in PC12 cells confirmed the retention of beta1 integrin, paxillin, talin, and vinculin with the cytoskeleton. Moreover, we detected FAK+ and RhoA in the detergent-resistant cytoskeleton, supporting their distribution to point contacts. Our observations indicate that two types of integrin clusters are present in growth cones: those associated with vinculin at the cell substratum interface, and those not associated with vinculin. Point contacts are mature adhesion sites defined by the presence of both beta1 integrin and vinculin, and they are associated with signaling proteins.

Conversion of neuronal growth cone responses from repulsion to attraction by cyclic nucleotides.

Nerve growth is regulated by attractive and repulsive factors in the nervous system. Microscopic gradients of Collapsin-1/Semaphorin III/D (Sema III) and myelin-associated glycoprotein trigger repulsive turning responses by growth cones of cultured Xenopus spinal neurons; the repulsion can be converted to attraction by pharmacological activation of the guanosine 3',5'-monophosphate (cGMP) and adenosine 3',5'-monophosphate signaling pathways, respectively. Sema III also causes the collapse of cultured rat sensory growth cones, which can be inhibited by activation of the cGMP pathway. Thus cyclic nucleotides can regulate growth cone behaviors and may be targets for designing treatments to alleviate the inhibition of nerve regeneration by repulsive factors.

Unique responses of differentiating neuronal growth cones to inhibitory cues presented by oligodendrocytes.

During central nervous system development, neurons differentiate distinct axonal and dendritic processes whose outgrowth is influenced by environmental cues. Given the known intrinsic differences between axons and dendrites and that little is known about the response of dendrites to inhibitory cues, we tested the hypothesis that outgrowth of differentiating axons and dendrites of hippocampal neurons is differentially influenced by inhibitory environmental cues. A sensitive growth cone behavior assay was used to assess responses of differentiating axonal and dendritic growth cones to oligodendrocytes and oligodendrocyte- derived, myelin-associated glycoprotein (MAG). We report that >90% of axonal growth cones collapsed after contact with oligodendrocytes. None of the encounters between differentiating, MAP-2 positive dendritic growth cones and oligodendrocytes resulted in growth cone collapse. The insensitivity of differentiating dendritic growth cones appears to be acquired since they develop from minor processes whose growth cones are inhibited (nearly 70% collapse) by contact with oligodendrocytes. Recombinant MAG(rMAG)-coated beads caused collapse of 72% of axonal growth cones but only 29% of differentiating dendritic growth cones. Unlike their response to contact with oligodendrocytes, few growth cones of minor processes were inhibited by rMAG-coated beads (20% collapsed). These results reveal the capability of differentiating growth cones of the same neuron to partition the complex molecular terrain they navigate by generating unique responses to particular inhibitory environmental cues.

Expression of specific tubulin isotypes increases during regeneration of injured CNS neurons, but not after the application of brain-derived neurotrophic factor (BDNF).

Axonal regrowth after injury is accompanied by changes in the expression of tubulin, but the contributions of substrate molecules and neurotrophic factors in regulating these changes in vivo are not known. Adult rat retinal ganglion cells (RGCs) were examined after intraorbital axotomy, after application of a peripheral nerve (PN) graft to stimulate regeneration, and after axotomy and treatment with brain-derived neurotrophic factor (BDNF). After these treatments we used in situ hybridization to study mRNA levels for betaI, betaII, betaIII, betaIVa, and Talpha1 tubulin isotypes. Levels of mRNA for all isotypes were downregulated after intraorbital axotomy. During regrowth of injured RGC axons, mRNA levels for betaII, betaIII, and Talpha1 isotypes were upregulated specifically and dramatically, suggesting that elevated expression of these isotypes is correlated specifically with axonal regrowth. A corresponding increase in betaIII protein levels was detected by immunocytochemistry. The betaI and betaIVa mRNAs were not increased during regeneration. BDNF did not elicit a specific increase in the mRNA levels for the betaIII and Talpha1 isotypes and had only a small effect on mRNA levels for the betaII isotype. Therefore, despite the ability of BDNF to support the survival of injured RGCs and to enhance neurite outgrowth of retinal neurons in vitro, the in vivo application of BDNF alone is unable to induce the program of changes in growth-associated tubulins that accompany regeneration of RGC axons into PN grafts. We speculate that, in addition to BDNF, cooperative signaling with substrate molecules is required to allow RGCs to regenerate and exhibit tubulin isotype switching.

Brain-derived neurotrophic factor modulates GAP-43 but not T alpha1 expression in injured retinal ganglion cells of adult rats.

The administration of neurotrophins affects neuronal survival and growth, but less is known about their ability to modify the expression of growth associated genes following injury to CNS neurons. Here we characterize the effect of brain-derived neurotrophic factor (BDNF) on mRNA levels for T alpha1 alpha-tubulin, and for GAP-43, two genes whose expression levels in retinal ganglion cells (RGC) tend to correlate with growth. We first determined that most adult rat RGCs can retrogradely transport BDNF by injecting 125I-BDNF into RGC target sites in vivo. We then used quantitative in situ hybridization to characterize the effect of axotomy, or axotomy and BDNF administration on mRNA levels for GAP-43 and T alpha1. Axotomy alone resulted in a general decrease in T alpha1 alpha-tubulin mRNA levels by 2 weeks, and elicited an increase in GAP-43 mRNA levels in an average of 30% of surviving RGCs. The intravitreal administration of a single dose of BDNF (5 microg) to axotomized RGCs on the day of injury did not affect T alpha1 alpha-tubulin mRNA levels, but was followed by a moderate (approximately 80%), and short-lasting enhancement of GAP-43 mRNA levels in most RGCs during the first week after axotomy. No significant increase in GAP-43 mRNA levels was observed when BDNF was injected into the uninjured eye. We conclude that BDNF specifically enhances GAP-43 but not T alpha1 mRNA levels in injured RGCs. Because BDNF is known to stimulate branch length of injured RGCs, we suggest that changes in the expression of GAP-43, but not T alpha1 tubulin, correlate with branching of injured neurons as opposed to long distance regrowth.

Myelin-associated glycoprotein inhibits neurite/axon growth and causes growth cone collapse.

We have previously shown that myelin-associated glycoprotein (MAG) inhibits neurite growth from a neuronal cell line. In this study we show that 60% of axonal growth cones of postnatal day 1 hippocampal neurons collapsed when they encountered polystyrene beads coated with recombinant MAG (rMAG). Such collapse was not observed with denatured rMAG. Neurite growth from rat embryonic hippocampal and neonatal cerebellar neurons was also inhibited about 80% on tissue culture substrates coated with rMAG. To investigate further the inhibitory activity of MAG in myelin, we purified myelin from MAG-deficient mice and separated octylglucoside extracts of myelin by diethylaminoethyl (DEAE) ion-exchange chromatography. Although there was no significant difference in neurite growth on myelin purified from MAG-/- and MAG+/+ mice, differences were observed in the fractionated material. The major inhibitory peak that is associated with MAG in normal mice was significantly reduced in MAG-deficient mice. These results suggest that although MAG contributes significantly to axon growth inhibition associated with myelin, its lack in MAG-deficient mice is masked by other non-MAG inhibitors. Axon regeneration in these mice was also examined after thoracic lesions of the corticospinal tracts. A very small number of anterogradely labeled axons extended up to 13.2 mm past the lesion in MAG-/- mice. Although there is some enhancement of axon generation, the poor growth after spinal cord injury in MAG-/- mice may be due to the presence of other non-MAG inhibitors. The in vitro studies, however, provide the first evidence that MAG modulates growth cone behavior and inhibits neurite growth by causing growth cone collapse.

Role of laminin and integrin interactions in growth cone guidance.

Laminin is well known to promote neuronal adhesion and axonal growth, but recent experiments suggest laminin has a wider role in guiding axons, both in development and regeneration. In vitro experiments demonstrate that laminin can alter the rate and direction of axonal growth, even when growth cone contact with laminin is transient. Investigations focused on a single neuronal type, such as retinal ganglion cells (RGCs), strongly implicate laminin as an important guidance molecule in development and suggest the involvement of integrins. Integrins are receptors for laminin, and neurons express multiple types of integrins that bind laminin. Morphologically, integrins cluster in point contacts, specialized regions of the growth cone that may coordinately regulate adhesion and motility. Recent evidence suggests that the structure and regulation of point contacts may differ from that of their nonneuronal counterpart, focal contacts. In part, this may be because the interaction of the cytoplasmic domain of integrin with the cytoskeleton is different in point contacts and focal contracts. Mutational studies where the cytoplasmic domain is truncated or altered are leading to a better understanding of the role of the alpha and beta subunit in regulating integrin clustering and binding to the cytoskeleton. In addition, whereas integrins may regulate motility through direct physical linkages to the growth cone cytoskeleton, an equally important role is their ability to elicit signaling, both through protein tyrosine phosphorylation and modulating calcium levels. Through such mechanisms integrins likely regulate the dynamic attachment and detachment of the growth cone as it moves on laminin substrates.

Laminin overrides the inhibitory effects of peripheral nervous system and central nervous system myelin-derived inhibitors of neurite growth.

Axon growth inhibitory proteins associated with central nervous system (CNS) myelin are responsible in part for the absence of long distance axon regeneration in the adult mammalian CNS. We have recently reported that myelin-associated glycoprotein (MAG), which is also present in peripheral nerves, is a potent inhibitor of neurite growth. This was surprising given the robust regenerative capacity of peripheral nerves. We now provide evidence that myelin purified from peripheral nerve also has neurite growth inhibitory activity. However, this activity can be masked by laminin, which is a constituent of the Schwann cell basal lamina. We also report that laminin, which is largely absent from the normal adult mammalian CNS, when added to purified CNS myelin, can override the neurite growth inhibitory activity in CNS myelin. These results have important implications for the development of strategies to foster axon regeneration in the adult mammalian CNS where multiple growth inhibitors exist.

Tubulin expression and axonal transport in injured and regenerating neurons in the adult mammalian central nervous system.

Microtubules are essential components of the cytoskeleton required for axonal growth. To investigate how changes in tubulin transport and expression may affect axon regeneration, injury in the adult mammalian central nervous system was studied. Axotomized retinal ganglion cells (RGCs) that do not regenerate were compared with RGCs that regenerate their axons when the optic nerve is replaced with a peripheral nerve graft. When RGC axons regenerated through peripheral nerve grafts, the rate of slow transport increased but decreased when no regrowth occurred. To investigate the molecular mechanisms that mediate these responses, alterations in tubulin mRNA levels after injury were examined. Total tubulin mRNA levels fell after injury in the optic nerve but increased in those RGCs that regenerated their axons into a peripheral nerve graft. Further, the expression of four separate beta-tubulin isotypes in injured rat RGCs was characterized. mRNA levels for all four isotypes decreased in RGCs after injury in the optic nerve. How the autoregulation of tubulin expression may contribute to the changes in beta-tubulin isotype expression after injury is discussed.