Best practice guidelines for stroke in Cameroon: An innovative and participatory knowledge translation project

Background: Although the adherence to stroke guidelines in high-income countries has been shown to be associated with improved patient outcomes, the research, development and implementation of rehabilitation related guidelines in African countries is lacking. Objectives: The purpose of this article is to describe how a group of front-line practitioners collaborated with academics and students to develop best practice guidelines (BPG) for the management and rehabilitation of stroke in adult patients in Cameroon. Method: A working group was established and adapted internationally recognised processes for the development of best practice guidelines. The group determined the scope of the guidelines, documented current practices, and critically appraised evidence to develop guidelines relevant to the Cameroon context. Results: The primary result of this project is best practice guidelines which provided an overview of the provision of stroke rehabilitation services in the region, and made 83 practice recommendations to improve these services. We also report on the successes and challenges encountered during the process, and the working group’s recommendations aimed at encouraging others to consider similar projects. Conclusion: This project demonstrated that there is interest and capacity for improving stroke rehabilitation practices and for stroke guideline development in Africa

Evidence of severe mitochondrial oxidative stress and a protective effect of low oxygen in mouse models of inherited photoreceptor degeneration

The role of oxidative stress within photoreceptors (PRs) in inherited photoreceptor degeneration (IPD) is unclear. We investigated this question using four IPD mouse models (Pde6b(rd1/rd1), Pde6b(atrd1/atrd1), Rho(-/-) and Prph2(rds/rds)) and compared the abundance of reduced glutathione (GSH) and the activity of mitochondrial NADH:ubiquinone oxidoreductase (complex I), which is oxidative stress sensitive, as indirect measures of redox status, in the retinas of wild type and IPD mice. All four IPD mutants had significantly reduced retinal complex I activities (14-29% of wild type) and two showed reduced GSH, at a stage prior to the occurrence of significant cell death, whereas mitochondrial citrate synthase, which is oxidative stress insensitive, was unchanged. We orally administered the mitochondrially targeted anti oxidant MitoQ in order to reduce oxidative stress but without any improvement in retinal complex I activity, GSH or rates of PR degeneration. One possible source of oxidative stress in IPDs is oxygen toxicity in the outer retina due to reduced consumption by PR mitochondria. We therefore asked whether a reduction in the ambient O-2 concentration might improve PR survival in Pde6b(rd1/rd1) retinal explants either directly, by reducing reactive oxygen species formation, or indirectly by a neuroprotective mechanism. Pde6b(rd1/rd1) retinal explants cultured in 6% O-2 showed 31% less PR death than normoxic explants. We conclude that (i) mitochondrial oxidative stress is a significant early feature of IPDs; (ii) the ineffectiveness of MitoQ may indicate its inability to reduce some mediators of oxidative stress, such as hydrogen peroxide; and (iii) elucidation of the mechanisms by which hypoxia protects mutant PRs may identify novel neuroprotective pathways in the retina

Endothelin-2-Mediated Protection of Mutant Photoreceptors in Inherited Photoreceptor Degeneration

<div><p>Expression of the <i>Endothelin-2 (Edn2)</i> mRNA is greatly increased in the photoreceptors (PRs) of mouse models of inherited PR degeneration (IPD). To examine the role of <i>Edn2</i> in mutant PR survival, we generated <i>Edn2^−/−</i> mice carrying homozygous <i>Pde6b^rd1</i> alleles or the <i>Tg(RHO P347S)</i> transgene. In the <i>Edn2^−/−</i> background, PR survival increased 110% in <i>Pde6b^rd1/rd1</i> mice at post-natal (PN) day 15, and 60% in <i>Tg(RHO P347S)</i> mice at PN40. In contrast, PR survival was not increased in retinal explants of <i>Pde6b^rd1/rd1</i>; <i>Edn2^−/−</i> mice. This finding, together with systemic abnormalities in <i>Edn2^−/−</i> mice, suggested that the increased survival of mutant PRs in the <i>Edn2^−/−</i> background resulted at least partly from the systemic EDN2 loss of function. To examine directly the role of EDN2 in mutant PRs, we used a scAAV5-<i>Edn2</i> cDNA vector to restore <i>Edn2</i> expression in <i>Pde6b^rd1/rd1</i>; <i>Edn2^−/−</i> PRs and observed an 18% increase in PR survival at PN14. Importantly, PR survival was also increased after injection of scAAV5-<i>Edn2</i> into <i>Pde6b^rd1/rd1</i> retinas, by 31% at PN15. Together, these findings suggest that increased <i>Edn2</i> expression is protective to mutant PRs. To begin to elucidate <i>Edn2</i>-mediated mechanisms that contribute to PR survival, we used microarray analysis and identified a cohort of 20 genes with >4-fold increased expression in <i>Tg(RHO P347S)</i> retinas, including <i>Fgf2</i>. Notably, increased expression of the FGF2 protein in <i>Tg(RHO P347S)</i> PRs was ablated in <i>Tg(RHO P347S)</i>; <i>Edn2^−/−</i> retinas. Our findings indicate that the increased expression of PR <i>Edn2</i> increases PR survival, and suggest that the <i>Edn2</i>-dependent increase in PR expression of FGF2 may contribute to the augmented survival.</p> </div

Retinal <i>Edn2</i> mRNA is increased in several models of IPD.

<p>(A) qRT-PCR assays of the <i>Edn2</i> mRNA. Edn2 was increased 32-fold, 70-fold, and 72-fold in the <i>Prph2^rds/+</i> (7 weeks), <i>Tg(RHO P347S)</i> (3 weeks) and <i>Pde6b^rd1/rd1</i> (PN12) models of IPD, respectively (^**n = 3; Student's t-test p<0.005). At the three time points chosen, >40% of the PR population remained. <i>Edn2</i> mRNA expression was assigned a value of 1 in WT retinas, to calculate its relative expression in the IPD models. qRT-PCR values were normalized to the mRNA expression of <i>Gapdh</i>. (B) <i>In situ</i> hybridization of <i>Edn2</i> mRNA in <i>Prph2^rds/+</i> and <i>Tg(RHO P347S)</i> retinas. <i>Edn2</i> transcripts were undetectable in WT retina, but were detected exclusively in the ONL of the mutant PRs (arrowheads) in <i>Prph2^rds/+</i> and <i>Tg(RHO P347S)</i> retinas. (C,D) Temporal expression of <i>Edn2</i> transcripts during PR degeneration in <i>Pde6b^rd1/rd1</i> retinas, relative to WT <i>Edn2</i> mRNA expression at PN12 and normalized to <i>Gapdh</i> mRNA (C), or rhodopsin mRNA (D). <i>Edn2</i> mRNA was not significantly increased until PN10 (^*n = 3; p<0.05 vs. WT). RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Error bars indicate SEM; scale bar = 10 µm.</p

Systemic erythropoietin (EPO) and retinal vascular endothelial growth factor (VEGF) is increased in <i>Edn2^−/−</i> mice.

<p>At PN21, serum EPO was increased 11-fold in Edn2^−/− vs. Edn2^+/+ mice (n = 7; p<0.005) (A) and retinal VEGF was increased 4-fold (n = 4; p<0.005) (B). EPO and VEGF were both measured using ELISA assays. Error bars indicate SEM.</p

Expression of GFP and <i>Edn2</i> from subretinally injected scAAV5.

<p>(A) The temporal and spatial expression of GFP in WT retinas injected subretinally with 1X PBS or scAAV5-smCBA-<i>Gfp</i> at PN8 and evaluated at PN10, PN12, and PN14 by GFP immunofluorescence (A, Panels 1–4). Significant GFP staining was not observed until PN12, with stronger staining at PN14, especially in the vicinity of the subretinal injection site (arrow). The spatial expression of GFP in WT retinas injected with scAAV5-smCBA-<i>Gfp</i> was also evaluated in paraffin sections at PN12 (A, Panel 5). GFP expression was observed predominantly in the ONL and RPE; sporadic expression of GFP in Müller cells was also observed in paraffin sections. (Bar = 25 µm.) (B) Schematic of the EDN2 cleavage events required to produce the mature EDN2 peptide. EDN2 is first produced as prepro EDN2 (175 aa) which is rapidly processed by furin-like endopeptidases to yield big EDN2 (38 aa). Big EDN2 must then be cleaved by an endothelin-specific converting enzyme (ECE) to produce the 21 a.a. mature EDN2 peptide that can bind to endothelin receptors (figure adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058023#pone.0058023-Saida1" target="_blank">[68]</a>). The regions of the <i>Edn2</i> mRNA corresponding to the cDNAs cloned into the scAAV5-<i>smCBA</i>-prepro<i>Edn2</i> and scAAV5-<i>smCBA</i>-mat<i>Edn2</i> vectors are shown. (C) Expression of scAAV5-derived <i>Edn2</i> mRNA in <i>Pde6b^rd1/rd1</i> retinas at PN12 after injection of the scAAV5-<i>smCBA</i>-prepro<i>Edn2</i> and scAAV5-<i>smCBA</i>-mat<i>Edn2</i> constructs at PN8. scAAV5-derived <i>Edn2</i> mRNA expression values are shown relative to the levels of endogenous <i>Edn2</i> mRNA (from the same retina) and all values were normalized to <i>Gapdh</i>. scAAV5-prepro<i>Edn2</i> transcripts were increased between 1.7 and 7.2-fold (n = 4; average 4.3-fold) over endogenous <i>Edn2</i> mRNA, while scAAV5-mat<i>Edn2</i> transcripts increased between 2.5 to 11.3-fold over the endogenous <i>Edn2</i> mRNA (n = 4; average 6.9-fold). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Error bars indicate SEM.</p

scAAV5-mediated transfer of the mature <i>Edn2</i> cDNA at PN8 increases mutant PR survival.

<p>(A) Micrographs showing ONL thickness in individual retinas at PN14–15. (a) Injection of the scAAV5-<i>smCBA</i>-mat<i>Edn2</i> vector into WT retinas at PN8 did not alter retinal morphology at PN15. (b) Injection of the scAAV5-<i>smCBA</i>-mat<i>Edn2</i> vector at PN8 increased PR ONL thickness of <i>Pde6b^rd1/rd1</i> retinas by 31% (n = 9; ^*p<0.05) at PN15, and (c) of <i>Pde6b^rd1/rd1</i>; <i>Edn2^−/−</i> retinas at PN14 by 18% (n = 5; ^*p<0.05). (d) In contrast, injection of the scAAV5-<i>smCBA</i>-prepro<i>Edn2</i> vector at PN8 had no effect on PR ONL thickness of <i>Pde6b^rd1/rd1</i> retinas (n = 6;p>0.05) at PN15, although (e) this vector improved PR survival in <i>Pde6b^rd1/rd1</i>; <i>Edn2^−/−</i> retinas by 14% (n = 6; ^*p<0.05). (B) A bar graph summarizing the effects of AAV vectors expressing <i>Edn2</i> cDNAs, injected at PN8, on mutant PR survival in <i>Pde6b^rd1/rd1</i> and <i>Pde6b^rd1/rd1</i>; <i>Edn2^−/−</i> retinas at PN14–15. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (Black bar = 25 µm). Error bars indicate SEM.</p